Methods of diagnosis, selection, and treatment of diseases and conditions caused by or associated with methanogens

ABSTRACT

The invention described herein provides for methods and systems for determining, selecting, and/or treating diseases and conditions caused by or associated with high quantities of methanogens in a subject, or diseases and conditions caused by or associated with low quantities of methanogens in a subject. In various embodiments, a therapy to inhibit the growth of methanogens or to promote the growth of methanogens are selected and/or administered to a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 121 asa divisional of U.S. patent application Ser. No. 14/211,197, filed Mar.14, 2014, which also claims priority to 35 U.S.C. § 119(e) to U.S.provisional patent application No. 61/792,687, filed Mar. 15, 2013, nowexpired, the entirety of which is incorporated herein by reference.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

The human gastrointestinal (GI) tract is host to a vast number ofmicroorganisms, which include archaea, bacteria, and eukaryotes. Todate, at least 70 divisions of bacteria and 13 divisions of archaea havebeen identified, and their collective genome (the microbiome) isbelieved to contain 100 times more genes than the human genome (A1,A2).Although the composition and number of microbes in the gut depends onmany factors (A3,A4), by adulthood most humans reach an established,relatively stable balance of type, and numbers of microbes that isunique to a given individual (A5). This microbial community is thoughtto develop with the host by establishing symbiotic relationships whichfavor their coexistence (A3,A6), such as assisting the host in thebreakdown of food for absorption and elimination (A7). While the fullbreadth of the impact of these gut microbes on the human host will takeyears to uncover, the complex and often interdependent relationshipsbetween gut microbes and the human host have been of increasingscientific interest this past decade, and this interest continues togrow. In particular, there is ample and growing evidence to suggestpotential roles for gut microbes in energy homeostasis, inflammation,and insulin resistance (A8-A10), and as a result, gut microbes have beenconsidered as possible causative factors of metabolic conditions andobesity, as well as potential therapeutic targets (A11-A15).

Methanogens are important constituents of gut microbiota that colonizethe human intestinal tract. These organisms are not bacteria but archaeaand generate methane by utilizing hydrogen and carbon dioxide (fromsyntrophic hydrogen producing bacteria) [10]. Several decades ago,Miller and Wolin isolated methanogens which were morphologically andphysiologically similar to Methanobrevibacter smithii from fecalspecimens of nine adults demonstrating methane production by enrichmentcultures. When examined by immunological methods, these isolates werevery closely related to M. smithii and unrelated or poorly related toother members of the Methanobacteriaceae family [11]. Utilizing the samemorphological and immunological techniques, Weaver et al detected M.smithii in tap water enema samples of 70% of their subjects beforesigmoidoscopy. A small subset of these patients who underwent breathanalysis needed at least 2×10⁸ methanogens/gm dry weight of stool tohave detectable breath methane of >6 parts per million (ppm) [12].However, these studies have not examined subjects with IBS and have notbeen replicated using molecular techniques such as PCR.

This distinct group grows primarily under anaerobic conditions, andproduces methane (CH₄) as a byproduct of fermentation. Methanogens areunique in that their metabolism increases in the presence of productsfrom other gut microbes (A16), as they scavenge hydrogen and ammonia assubstrates for the generation of methane (A17,A18). Once absorbed intosystemic circulation, methane is cleared via the lungs. The majority ofmethanogens found in the human gut are from the genusMethanobrevibacter; predominantly Methanobrevibacter smithii (A7). M.smithii is found in 70% of human subjects, and analysis of expiratorymethane by lactulose breath testing can serve as an indirect measure ofmethane production (A7,A19). A minority of subjects (15%) produce largequantities of methane early in the breath test, suggesting a greatermethane potential (A20), and increased methane production on breath testcorrelates with increased levels of M. smithii in stool, as determinedby quantitative PCR (qPCR) (A20,A21).

Introduction of both a Bacteroides species (Bacteroidesthetaiotaomicron) and M. smithii into germ-free mice resulted in greaterbody weights than with B. thetaiotaomicron alone (A22), and methanogenshave been shown to increase the capacity of polysaccharide-metabolizingbacteria to digest polyfructose-containing glycans in the colons ofgerm-free mice (A22), suggesting that methanogens may play a role incaloric harvest. In humans, the inventors have recently found thatincreased methane on breath test is associated with a higher averageBMI, both in normal population and in obese subjects. In the obesepopulation, methane was associated with a remarkable 6.7 kg/m2 greaterBMI compared to non-methane controls (P<0.05) (A23). While these dataare suggestive of a role for methanogens in caloric harvest and weightgain in humans, this is weakened by the fact that, to date, colonizationwith methanogens has only been demonstrated in the large bowel(A24-A26).

Therefore, as described herein the inventors tested and compared weightgain and the location and extent of M. smithii colonization in the GItracts of rats under different dietary conditions. Also describedherein, the inventors examine the importance of Methanobrevibactersmithii as a determinant of methane production in the breath of humansusing quantitative-polymerase chain reaction (PCR) from stool of IBSpatients with and without detectable methane on breath testing.

Obesity constitutes a significant and rapidly increasing public healthchallenge and is associated with increased risks for coronary arterydisease, hypertension, stroke, type 2 diabetes, certain cancers, andpremature death (B1, B2). Elucidating mechanisms contributing to thedevelopment of obesity is central to defining preventive approaches.Research has begun to define the relationship between gut flora andmetabolism (B3-B5). Alterations in the relative abundance ofBacteroidetes and Firmicutes have been linked to changes in metabolismand weight increases both in mice (B6) and humans (B4). Cocolonizationwith the methanogenic archaea, Methanobrevibacter smithii, results in agreater weight gain in germ-free animals than infection with Bthetaiotaomicron alone (B7).

Accordingly, there exists a need for methods for determining thepresence of methanogens, and their cause and/or association with variousdiseases and conditions, and selecting and/or administering anappropriate treatment for those diseases and conditions, such asobesity, pre-diabetes diabetes, diabetes, insulin resistance, glucoseintolerance, constipation, fatty liver, Crohn's disease and ulcerativecolitis, to name a few.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with compositions and methods which are meantto be exemplary and illustrative, not limiting in scope.

Various embodiments provide for a method, comprising subjecting abiological sample from a subject to analysis for methanogen quantity;comparing the methanogen quantity to a reference value; and selecting afirst therapy for the subject if the methanogen quantity is higher thanthe reference value based on the recognition that the first therapy isappropriate for subjects who have a methanogen quantity higher than thereference value, or selecting a second therapy for the subject if themethanogen quantity is lower than the reference value based on therecognition that the second therapy is appropriate for subjects who havea methanogen quantity lower than the reference value, wherein thesubject has or is suspected to have a disease or condition caused by orassociated with having a high methanogen quantity or a disease orcondition caused by or associated with having a low methanogen quantity.

Various embodiments of the present invention provide for a method,comprising subjecting a biological sample from a subject to analysis formethanogen quantity; comparing the methanogen quantity to a referencevalue; and selecting a first therapy for the subject if the methanogenquantity is higher than the reference value based on the recognitionthat the first therapy is appropriate for subjects who have a methanogenquantity higher than the reference value, or selecting a second therapyfor the subject if the methanogen quantity is lower than the referencevalue based on the recognition that the second therapy is appropriatefor subjects who have a methanogen quantity lower than the referencevalue, wherein the subject desires a determination of susceptibility tohaving a disease or condition caused by or associated with having a highmethanogen quantity or a disease or condition caused by or associatedwith having a low methanogen quantity.

In various embodiments, the method can further comprise providing thebiological sample. In various embodiments, the method can furthercomprise administering the selected therapy.

In various embodiments, the method can further comprise subjecting thebiological sample to analysis for a quantity of a methanogen syntrophicmicroorganism. In various embodiments, the methanogen syntrophicmicroorganism can be a hydrogen-producing microorganism. In variousembodiments, the method can further comprise selecting a third therapyto inhibit the growth of the methanogen syntrophic microorganism. Invarious embodiments, the method can further comprise administering thethird therapy.

In various embodiments of the method, the biological sample can beselected from stool, mucosal biopsy from a site in the gastrointestinaltract, aspirated liquid from a site in the gastrointestinal tract, orcombinations thereof. In various embodiments of the method, the site inthe gastrointestinal tract can be mouth, stomach, small intestine, largeintestine, anus or combinations thereof. In various embodiments of themethod, the site in the gastrointestinal tract can be duodenum, jejunum,ileum, or combinations thereof. In various embodiments of the method,the site in the gastrointestinal tract can be cecum, colon, rectum, anusor combinations thereof. In various embodiments of the method, the sitein the gastrointestinal tract can be ascending colon, transverse colon,descending colon, sigmoid flexure, or combinations thereof.

In various embodiments of the method, the analysis for methanogenquantity can be by using quantitative polymerase chain reaction (qPCR).

In various embodiments of the method, the disease or condition caused byor associated with having the high methanogen quantity can be selectedfrom the group consisting of obesity, constipation, fatty liver (NASH),pre-diabetes, diabetes, insulin resistance, glucose intolerance andcombinations thereof.

In various embodiments of the method, the disease or condition caused byor associated with having the low methanogen quantity can be Crohn'sdisease or ulcerative colitis.

In various embodiments of the method, the methanogen can be from thegenus Methanobrevibacter. In various embodiments, the Methanobrevibactercan be selected from the group consisting of M. acididurans, M.arboriphilus, M. curvatus, M. cuticularis, M. filiformis, M.gottschalkii, M. millerae, M. olleyae, M. oralis, M. ruminantium, M.smithii, M. thaueri, M. woesei, M. wolinii and combinations thereof. Invarious embodiments, the Methanobrevibacter can be Methanobrevibactersmithii (M. Smithii).

In various embodiments of the method, the reference value can be about10,000 per ml of the biological sample.

In various embodiments of the method, the first therapy can be anantibiotic or a combination of two or more antibiotics. In variousembodiments, the antibiotic or the combination of two or moreantibiotics can be selected from the group consisting of rifaximin,neomycin, vancomycin, and metronidazole. In various embodiments, theantibiotic can be rifaximin. In various embodiments, the antibiotic canbe neomycin. In various embodiments, the antibiotic can be vancomycin.In various embodiments, the antibiotic can be metronidazole. In variousembodiments, the combination of two or more antibiotics can be rifaximinand neomycin, or rifaximin and metronidazole.

In various embodiments of the method, the first therapy can be aprobiotic capable of inhibiting the methanogen growth.

In various embodiments of the method, the first therapy can be areduced-calorie diet.

In various embodiments of the method, the first therapy can be areduced-fat diet.

In various embodiments of the method, the first therapy can be anelemental diet.

In various embodiments of the method, the first therapy can be a statin.In various embodiments, the statin can be selected from the groupconsisting of atorvastatin, fluvastatin, lovastatin, pitavastatin,pravastatin, rosuvastatin, simvastatin, and combinations thereof.

In various embodiments of the method, the disease or condition isobesity and the first therapy can be an anti-obesity drug. In variousembodiments, the anti-obesity drug can be phentermine,phentermine/topiramate, xenical, lorcaserin, or rimonabant.

In various embodiments of the method, the disease or a condition can beselected from the group consisting of pre-diabetes, diabetes, insulinresistance, and glucose intolerance, and the first therapy can beselected from the group consisting of alpha-glucosidase inhibitors,amylin analog, dipeptidyl peptidase-4 inhibitor, GLP1 agonist,meglitinide, sulfonylurea, biguanide, thiazolidinedione (TZD), insulin,and combinations thereof. In various embodiments, the alpha-glucosidaseinhibitors can be select from the group consisting of acarbose, miglitoland combinations thereof. In various embodiments, the amylin analog canbe pramlintide. In various embodiments, the dipeptidyl peptidase-4inhibitor can be selected from the group consisting of Saxagliptin,Sitagliptin, Vildagliptin, Linagliptin, Alogliptin, or combinationsthereof. In various embodiments, the GLP1 agonist can be selected fromthe group consisting of liraglutide exenatide, exenatide extendedrelease, or combinations thereof. In various embodiments, themeglitinide can be selected from the group consisting of nateglinide,repaglinide, and combinations thereof. In various embodiments, thesulfonylurea can be selected from the group consisting ofchlorpropamide, Glimepiride, Glipizide, Glyburide, Tolazamide,Tolbutamide and combinations thereof. In various embodiments, thebiguanide can be selected from the group consisting of Metformin,Riomet, Glucophage, Glucophage extended release, Glumetza, andcombinations thereof. In various embodiments, the thiazolidinedione canbe selected from the group consisting of Rosiglitazone, Pioglitazone andcombinations thereof. In various embodiments, the insulin can beselected from the group consisting of Aspart, Detemir, Glargine,Glulisine, Lispro, and combinations thereof.

In various embodiments of the method, the disease or a condition can beselected from the group consisting of pre-diabetes, diabetes, insulinresistance, and glucose intolerance, and the first therapy can beselected from the group consisting Glipizide/Metformin,Glyburide/Metformin, Pioglitazone/Glimepiride, Pioglitazone/Metformin,Repaglinide/Metformin, Rosiglitazone/Glimepiride,Rosiglitazone/Metformin, Saxagliptin/Metformin, Sitagliptin/Simvastatin,Sitagliptin/Metformin, Linagliptin/Metformin, Alogliptin/Metformin,Alogliptin/Pioglitazone, bromocriptine, welchol, and combinationsthereof.

In various embodiments of the method, the disease or condition can beconstipation, and the first therapy can be selected from the groupconsisting of laxative, diet, guanylate cyclase C agonist, a serotoninagonist, and combinations thereof. In various embodiments, the guanylatecyclase C agonist can be linaclotide. In various embodiments, theserotonin agonist can be prucalorpride, tegaserod or combinationsthereof.

In various embodiments of the method, the disease or condition can befatty liver, and the first therapy can be metformin.

In various embodiments of the method, the disease or condition can beCrohn's disease and the second therapy can be administering amethanogen.

In various embodiments, the methanogen can be from the genusMethanobrevibacter. In various embodiments, the Methanobrevibacter canbe selected from the group consisting of M. acididurans, M.arboriphilus, M. curvatus, M. cuticularis, M. filiformis, M.gottschalkii, M. millerae, M. olleyae, M. oralis, M. ruminantium, M.smithii, M. thaueri, M. woesei, M. wolinii and combinations thereof.

In various embodiment, the Methanobrevibacter can be Methanobrevibactersmithii (M. Smithii).

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts M. Smithii counts in methane and non-methane producers instool in accordance with various embodiments of the present invention.

FIG. 2 depicts percent M. smithii relative to prokaryotic bacteria asdeterminant of detection of methane on breath in accordance with variousembodiments of the present invention.

FIG. 3 depicts correlation between M. smithii and breath methane AUC inaccordance with various embodiments of the present invention.

FIG. 4 depicts correlation between percent M. smithii to totalprokaryotes and breath methane AUC in accordance with variousembodiments of the present invention.

FIG. 5 depicts correlation between hydrogen and methane in breath AUC inaccordance with various embodiments of the present invention.

FIG. 6 depicts relationship between M. smithii level and the relativedegree of constipation to diarrhea in accordance with variousembodiments of the present invention. C-D is a validated measure of therelative degree of constipation to diarrhea. The larger the number themore constipation is relative to diarrhea.

FIG. 7 depicts comparison of the percent M. smithii to total bacteria inthe stool and relative degree of constipation in accordance with variousembodiments of the present invention. C-D is a validated measure of therelative degree of constipation to diarrhea. The larger the number themore constipation is relative to diarrhea. The % M. smithii isdetermined by the amount of M. smithii relative to total prokaryoticbacteria.

FIG. 8 depicts correlation between total prokaryote bacteria counts instool and abdominal pain scores in accordance with various embodimentsof the present invention.

FIG. 9 depicts the effect of Methanobrevibacter smithii gavage on stoolquantity of this species over time (before diet manipulation). *P<0.01.in accordance with various embodiments of the present invention.

FIG. 10 depicts the effects of dietary fat content on rat weights andstool Methanobrevibacter smithii levels in accordance with variousembodiments of the present invention. (A) Rat weights over time startingfrom the adult weight plateau. *P<0.00001 change in weight after 1 weekon high-fat diet. ‡P<0.001 change in weight after return to highfatdiet. (B) Methanobrevibacter smithii levels over time. *P<0.01 forincrease in stool M. smithii after starting on high-fat chow. ⋄P<0.001for decrease in stool M. smithii after return to normal chow. †P=0.039for increase in stool M. smithii after return to high-fat chow.

FIG. 11 depicts the effect of high-fat diet on stool Methanobrevibactersmithii levels in accordance with various embodiments of the presentinvention. (A) Methanobrevibacter smithii levels before, 1 week after,and 5 weeks after high-fat diet. (B) Stool M. smithii levels and thedegree of weight gain. Comparing weight gain from day 98 to day 154. (C)Effect of returning to high-fat chow on stool M. smithii levels.

FIG. 12 depicts Methanobrevibacter smithii and total bacterial levels bysegment of bowel in accordance with various embodiments of the presentinvention. (A) Methanobrevibacter smithii by segment of bowelpost-mortem. P<0.001 between ileum and cecum and left colon; P=0.03comparing ileum to jejunum and P=0.07 comparing ileum to duodenum. (B)Total bacteria by segment of bowel post-mortem.

FIG. 13 depicts the effects of dietary fat content on Methanobrevibactersmithii and total bacterial levels in the bowel in accordance withvarious embodiments of the present invention. (A) Methanobrevibactersmithii throughout the bowel by diet. ♦P<0.05. (B) Total bacteriathroughout bowel by diet. None of the comparisons were significant.

FIG. 14 depicts the number of segments with no Methanobrevibactersmithii colonization and weight in accordance with various embodimentsof the present invention. Trend is not statistically significant.

FIG. 15 depicts body composition and production of methane and hydrogenon a breath test. (A) BMI by group. A significance level of P<0.02between the methane-and-hydrogen group and each of the other groups isshown. Error bars denote SEM. (B) Percent body fat by group. Asignificance level of P≤0.001 between the methane-and-hydrogen group andeach of the other groups is shown.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel,Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

“Beneficial results” may include, but are in no way limited to,lessening or alleviating the severity of the disease or condition,preventing or inhibiting the disease condition from worsening, curingthe disease condition and prolonging a patient's life or lifeexpectancy.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees, and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be including within the scope of this term.

“Therapeutically effective amount” as used herein refers to that amountwhich is capable of achieving beneficial results in a patient with ahigh quantity of methanogens or a patient with a low quantity ornonexistence of methanogens. A therapeutically effective amount can bedetermined on an individual basis and will be based, at least in part,on consideration of the physiological characteristics of the mammal, thetype of delivery system or therapeutic technique used and the time ofadministration relative to the progression of the disease or condition.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent, slow down and/or lessen the disease even if the treatmentis ultimately unsuccessful.

Described herein, the inventors demonstrated that Methanobrevibactersmithii is likely the important methanogen responsible for breathmethane in subjects with IBS. Furthermore, M. smithii levels andrelative proportions in stool correlate with the degree of methaneproduction suggesting this may be the major methanogen responsible formethane during breath testing in humans. Finally, this is the firststudy to demonstrate by qPCR that M. smithii is important in C-IBSsubjects with methane on LBT.

Recent literature suggests a role of methanogenic gastrointestinalmicrobiota in the pathophysiology of functional gastrointestinaldisorder such as IBS. Specifically, methane gas on LBT is associatedwith a constipation phenotype [5, 6, 7]. The inventors' group has shownthat methane is not an inert gas as previously thought; but slowsintestinal transit [14]. In an in-vivo study on 5 dogs, infusing methanethrough mid-small bowel fistula reduced proximal small bowel motility byan average of 59% [14]. The presence of breath methane has also beenassociated with significant slowing of intestinal transit in humanstudies [8, 15, 16]. Among patients with IBS, it has been confirmed in amultitude of publications that methane on lactulose breath testing isalmost universally associated with constipation predominant disease [5,6, 7, 8]. However, evaluation of stool in such patients in order todetermine the source of methane has never been attempted in IBS.

Described herein, the inventors established that Methanobrevibactersmithii is present ubiquitously in the stool of IBS patients. However,patients with methane-positive breath test harbor significantly greaterquantity of M. smithii as compared to the methane negative ones. Thesepatients also have higher proportions of M. smithii in their stoolrelative to other bacteria. The higher the count or relative proportionof M. smithii in stool, the greater the degree of breath methane. Thisimplies that stool quantitative-PCR is a much more sensitive tool thanbreath analysis in order to detect intestinal methanogens.

Interestingly, methanogens alone may not be problematic. In this study,most subjects had detectable M. smithii in their stool. However, thelevel of M. smithii may be the issue. Based on this study, methane onthe breath appears to be detectable when the level of M. smithii exceeds4.2×10⁵ copies per gm of wet stool or 1.2% of the total stool bacteria.This is important since in the original description of methane on breathand constipation IBS, not all constipation predominant IBS subjects hadmethane. However, nearly all methane subjects were constipated.Combined, these findings suggest that stool testing by qPCR may identifya threshold for producing constipation that a breath test is notsensitive enough to detect.

Also described herein, the threshold of M. smithii to cause detectablemethane on breath analysis was much smaller than that reported earlierby Weaver et al [12]. This difference is likely due to the use ofdiffering techniques. In the study by Weaver, et al, methanogens werecultured from the stool sample and identification as M. smithii wasbased on morphological and immunological methods. Handling and cultureof stool for methanogens can be difficult as the organisms areanaerobic. Exposure of the stool sample to air might harm the organismslimiting their growth. In the case of q-PCR, handling is not problematicsince PCR will detect both viable and non-viable organisms.

These data may have therapeutic and clinical significance as eliminationof methanogens by non-absorbable antibiotics can significantly improvegut symptoms [17, 18]. In methane producers with constipationpredominant IBS, neomycin resulted in 44.0±12.3% vs 5.0±5.1% improvementin constipation as compared to placebo that correlated well withelimination of methane on follow up breath testing [19]. In aretrospective study, combination of rifaximin and neomycin for 10 daysresulted in significantly greater reduction in methane (87%) andconstipation symptoms (85%) as compared to neomycin (33% and 63%,respectively) or rifaximin (28% and 56%, respectively) alone [20].

The inventors observed positive trends for association between M.smithii and constipation. The inventors' results suggest that M. smithiiis the predominant methanogenic archaeabacteria in the gut of C-IBSpatients responsible for methane on breath testing. This is supported bythe correlation between M. smithii level in stool and methane AUC onbreath testing.

Further described herein the inventors demonstrate for the first timethat colonization of the rat gut with the methanogen M. smithii is notlimited to the large bowel, but rather extends to the small bowel,including the ileum, jejunum, and duodenum. In fact, the inventors foundthat the levels of M. smithii were higher in the small bowel than in thelarge bowel, with the most elevated levels seen in the ileum. Moreover,the inventors found that switching rats to a high-fat diet resulted bothin increased levels of M. smithii in stool, and in increased levels ofM. smithii in all bowel segments tested. Most significantly, theinventors found that rats which gained more weight had higher stoollevels of M. smithii than rats which gained less weight, and that theextent of colonization of the bowel with M. smithii colonization alsocorresponded with weight gain in these rats, irrespective of diet. Takentogether, these findings support that the level and extent ofcolonization of the intestinal tract with M. smithii is predictive ofthe degree of weight gain in this animal model.

It is becoming increasingly understood that gut microbes play roles inand affect host metabolism and energy homeostasis, and the inventorsbelieve that they contribute to the development of metabolic disordersand obesity. Through the production of enzymes, gut microbes assist thehost in: utilizing nondigestible carbohydrates and host-derivedglycoconjugates, resulting in increased short-chain fatty acid (SCFA)production; deconjugating and dehydroxylating bile acids, which altersthe solubilization and absorption of dietary lipids; and cholesterolreduction and biosynthesis of vitamins from the K and B group,isoprenoids and amino acids such as lysine and threonine(A1,A11,A28,A29). Gut microbes have also been suggested to affectintestinal transit times, and to contribute to the chronic low-gradeinflammation and insulin resistance that are associated with obesity viaeffects on the endotoxin toll-like receptor 4 axis and intestinalbarrier function (A14,A30). These data support a role for gut microbesin contributing to weight gain by the host, which validates theinventors' finding that weight gain in the inventors' animal model wasmore dependent on the degree and extent of M. smithii colonization ofthe gut than on dietary fat content.

Several lines of evidence support that methanogens may play a specificrole in host metabolism and energy homeostasis. Methanogens such as M.smithii, which is the most common methanogen in the human gut, producemethane through anaerobic fermentation (A17,A18), and remove hydrogenatoms and accelerate the fermentation of polysaccharides andcarbohydrates (A22). This increases the production of SCFAs, which aresubsequently absorbed in the intestines and serve as an additionalenergy source for the host (A11). This more efficient energy extractionmay lead to weight gain and ultimately contribute to obesity (A32). Onepotential mechanism for this is through effects of SCFAs on Gprotein-coupled receptors, for which they act as ligands. The Gprotein-coupled receptor Gpr41 is expressed in the intestine, colon, andadipocytes, and stimulates the expression of the adipokine leptin andthe peptide tyrosine-tyrosine (peptide-YY), which both influence energymetabolism, and also affect appetite levels/satiety. In addition,modulation of plasma SCFAs has been linked to decreases of inflammatorymarkers in insulin-resistant human subjects (A33,A34), suggesting apotential effect on the chronic low-grade inflammation associated withobesity. Interestingly, in a recent human study the inventors found thatduring a 75 g oral glucose tolerance test, methane-producing subjects(i.e., those with increased methane on breath test) had greater serumglucose area-under-the-curve than non-methane subjects, despite havingcomparable BMIs and baseline insulin resistance (homeostatic modelassessment-insulin resistance), suggesting that intestinalmethane-producing subjects may have impaired glucose tolerance whenchallenged with a high carbohydrate load, and thus a highersusceptibility to hyperglycemia, than non-methane subjects (R. Mathur etal., data not shown). A final potential mechanism whereby methanogensmay affect energy extraction by the host is by slowing gut motility.Among human irritable bowel syndrome patients, the inventors found thatthose with methane on breath test are more likely to have constipationas a predominant symptom subtype (A19,A35), and that the amount ofmethane produced is related to the degree of constipation, as measuredby Bristol Stool Score, and frequency of bowel movements (A35). Methaneis also associated with other constipation disorders (A36,A37). In an invivo animal study, the inventors' group demonstrated that infusion ofmethane into the small intestine resulted in slowing of small intestinaltransit by 59% (A38). That slowing of intestinal transit may beassociated with greater BMI is demonstrated by a study by thegastroparesis consortium, which showed that subjects with extreme slowmotility (gastroparesis) had higher BMIs (A39), and by a study ofultrashort bowel patients, in which the inventors' group found thatslowing the gut with exenatide resulted in resolution of diarrhea,nutritional deficiencies and the need for chronic parenteral nutrition,and was accompanied by weight gain (A40). Taken together, theserepresent several potential mechanisms by which the increased M. smithiicolonization could contribute to the concomitant weight gain theinventors observed in these rats.

To date, methanogens have been identified primarily in the left colon(A24-A26), and it has been argued that alterations to a gut microbialpopulation not known to occur outside of the large bowel is unlikely tobe a significant direct cause of weight gain. The inventors' resultsdemonstrate for the first time that in the rat, not only doescolonization with M. smithii occur in the small bowel, but that M.smithii levels in the duodenum, jejunum, and ileum are in fact higherthan in the cecum or left colon, with highest levels in the ileum.Moreover, the degree of weight gain in these animals corresponded withthe number of bowel segments colonized, and the inventors believe thatthe extent of colonization of the intestine with M. smithii ispredictive of, and contribute to, weight gain.

In conclusion, the inventors' results demonstrate for the first timethat colonization with the methanogen M. smithii is not confined to thelarge intestine, but also occurs in the small bowel. Moreover, in thisrat model, the inventors found that the levels and extent of smallintestinal colonization with M. smithii correlated with, and werepredictive of, the degree of weight gain, irrespective of dietary fatcontent.

Also described herein, the inventors demonstrate clear associationsbetween the presence of both methane and hydrogen on breath testing andincreased BMI as well as increased percent body fat in an analysis ofnearly 800 subjects. This study is the first of its kind to identify theproduction of methane and hydrogen as an indicator of higher BMI and fatcontent in human subjects.

Obesity is a public health problem and is undoubtedly multifactorial.Dysregulations are seen in multiple areas of energy intake, expenditure,and storage. There is growing interest in the potential role of gutflora in the pathogenesis of obesity. Research by Gordon, Backhed, andothers (B3-B7) have shown an intriguing relationship between microbialflora and weight gain in mouse models, including an association betweenalterations in the relative abundance of Firmicutes vs Bacteroidetes inthe gut and potentially enhanced nutritional harvest (B3). Intestinalflora have been implicated in many mechanisms that may contribute toweight gain, including enhanced lipopolysaccharide production leading toinsulin resistance (B5), suppression of fasting-induced adipose factor(B14), suppression of AMP-activated protein kinase-driven fatty acidoxidation in the liver (B15), incretin regulation (B16), and increasedSCFA production and absorption, thereby providing increased lipogenicsubstrates to the host (B17). Increased methanogens have also beenobserved in the cecal flora of Ob/Ob mice (B3). The inventors believethat this large-scale human study described herein shows a role formethanogens, and specifically M smithii, in human obesity.

The human GI tract is colonized by up to 10¹² microbial species,including bacteria and archaea, of which M smithii is the most abundantmethane-producing organism (B9). The inventors show herein thatmethane-positive individuals have M smithii in the GI tract. Theinventors have shown that increased methane on breath testing isassociated with higher levels of M smithii in stool, and thatmethane-positive obese subjects have an average 6.7 kg/m² greater BMIthan methane-negative obese controls (B11). Although M smithii wasoriginally thought to inhabit only the large bowel, weakening thelikelihood that it could play a significant role in caloric harvest andweight gain, the inventors show herein using a rat model that M smithiicolonization in fact occurs throughout the small intestine. Importantly,the number of bowel segments colonized with M smithii was directlyrelated to the degree of weight gain in this rat model and was furtherenhanced in the presence of a high-fat diet.

The inventors believe that the role of M smithii in weight gain inanimals is facilitative and involves a syntrophic relationship withother microbes, whereby M smithii scavenges hydrogen produced bysyntrophic organisms for its hydrogen-requiring anaerobic metabolism,producing methane as a byproduct. This scavenging of hydrogen allows thesyntrophic organisms to be more productive, increasing SCFA productionand availability of calories for the host (B8). The inventors' resultssupport this—the presence of both hydrogen and methane on breath test,but not either methane or hydrogen alone, is associated with higher BMIand percent body fat, perhaps because these subjects have an abundanceof hydrogen to fuel methane production.

In addition, methane itself (in gaseous form as generated by intestinalmethanogens) could also contribute to enhanced energy harvest. Theinventors previously noted an association between breath methane andconstipation (constipation-irritable bowel syndrome) in human subjects(B13) and, using an in vivo animal model, demonstrated that methane gasdirectly slows transit in the gut by 59% (B19). The inventors believethat the slowing of transit could result in greater time to harvestnutrients and absorb calories, representing another potential mechanismfor weight gain.

Although the mean age of the methane producers was higher than that ofthe controls, the results retained significance, even when controllingfor age as a confounding variable. Furthermore, there is no evidence tosuggest that methane production increases with age but rather plateausin adulthood (B20), making it unlikely that age could affect the studyfindings. The inventors show herein that diet can affect overallintestinal flora and M smithii levels in animal models. The inventors'study does not account for dietary differences among subjects. However,given the large sample size, these individual variations may bemitigated between groups.

In summary, the inventors' study demonstrates for the first time thatindividuals with both methane and hydrogen on a breath test have higherBMIs and percent body fat. The inventors believe that this is due toexcessive colonization with the hydrogen-requiring methanogen M.smithii, which enhances energy harvest and delivery of nutrients to thehost organism through syntrophic relationships with other microbes.

Various embodiments of the present invention are based, at least inpart, on these findings.

Various embodiments of the present invention provide for a method forselecting and/or administering a therapy for a subject who has or issuspected to have a disease or condition caused by or associated withhaving a high methanogen quantity or a disease or condition caused by orassociated with having a low methanogen quantity. The method comprisessubjecting a biological sample from a subject to analysis for methanogenquantity; comparing the methanogen quantity to a reference value; andselecting a first therapy for the subject if the methanogen quantity ishigher than the reference value based on the recognition that the firsttherapy is appropriate for subjects who have a methanogen quantityhigher than the reference value, or selecting a second therapy for thesubject if the methanogen quantity is lower than the reference valuebased on the recognition that the second therapy is appropriate forsubjects who have a methanogen quantity lower than the reference value,wherein the subject has or is suspected to have a disease or conditioncaused by or associated with having a high methanogen quantity or adisease or condition caused by or associated with having a lowmethanogen quantity. First therapy and second therapy as used in thiscontext do not refer to administering two therapies, it is simply toprovide a distinctions between two types of therapies. The first therapyis a therapy that is appropriate for treating subjects who have highquantities of methanogens. The second therapy is a therapy that isappropriate for treatment subject who have low or non-detectablequantities of methanogens.

In various embodiments, the method further comprises identifying thesubject who has or is suspected to have a disease or condition caused byor associated with having a high methanogen quantity or a disease orcondition caused by or associated with having a low methanogen quantity.

In various embodiments, the method further comprises obtaining orproviding the biological sample. In various embodiments, the methodfurther comprises administering the selected therapy.

In various embodiments, the method further comprises subjecting thebiological sample to analysis for a quantity of a methanogen syntrophicmicroorganism. In various embodiments, the methanogen syntrophicmicroorganism is a hydrogen-producing microorganism. In variousembodiments, the method further comprises selecting a third therapy toinhibit the growth of the methanogen syntrophic microorganism. Invarious embodiments, the method further comprises administering thethird therapy. In various embodiments, the third therapy and the firsttherapy can be the same or same type of therapy.

Various embodiments provide for a method for selecting a therapy for asubject who desires a determination of susceptibility to having adisease or condition caused by or associated with having a highmethanogen quantity or a disease or condition caused by or associatedwith having a low methanogen quantity. The method comprises subjecting abiological sample from a subject to analysis for methanogen quantity;comparing the methanogen quantity to a reference value; and selecting afirst therapy for the subject if the methanogen quantity is higher thanthe reference value based on the recognition that the first therapy isappropriate for subjects who have a methanogen quantity higher than thereference value, or selecting a second therapy for the subject if themethanogen quantity is lower than the reference value based on therecognition that the second therapy is appropriate for subjects who havea methanogen quantity lower than the reference value, wherein thesubject desires a determination of susceptibility to having a disease orcondition caused by or associated with having a high methanogen quantityor a disease or condition caused by or associated with having a lowmethanogen quantity.

In various embodiments, the method further comprises identifying thesubject who desires a determination of susceptibility to having adisease or condition caused by or associated with having a highmethanogen quantity or a disease or condition caused by or associatedwith having a low methanogen quantity

In various embodiments, the method further comprises providing thebiological sample.

In various embodiments, the method further comprises administering theselected therapy.

In various embodiments, the method further comprises subjecting thebiological sample to analysis for a quantity of a methanogen syntrophicmicroorganism. In various embodiments, the methanogen syntrophicmicroorganism is a hydrogen-producing microorganism. In variousembodiments, the method further comprises selecting a third therapy toinhibit the growth of the methanogen syntrophic microorganism. Invarious embodiments, the method further comprises administering thethird therapy.

Various embodiments of the present invention provide for a method fortreating a subject who has or is suspected to have a disease orcondition caused by or associated with having a high methanogen quantityor a disease or condition caused by or associated with having a lowmethanogen quantity. The method comprises subjecting a biological samplefrom a subject to analysis for methanogen quantity; comparing themethanogen quantity to a reference value; selecting a first therapy forthe subject if the methanogen quantity is higher than the referencevalue based on the recognition that the first therapy is appropriate forsubjects who have a methanogen quantity higher than the reference value,or selecting a second therapy for the subject if the methanogen quantityis lower than the reference value based on the recognition that thesecond therapy is appropriate for subjects who have a methanogenquantity lower than the reference value; and administering the selectedtherapy to the patient to treat the disease or condition, wherein thesubject has or is suspected to have a disease or condition caused by orassociated with having a high methanogen quantity or a disease orcondition caused by or associated with having a low methanogen quantity.

First therapy and second therapy as used in this context do not refer toadministering two therapies, it is simply to provide a distinctionsbetween two types of therapies. The first therapy is a therapy that isappropriate for treating subjects who have high quantities ofmethanogens. The second therapy is a therapy that is appropriate fortreatment subject who have low or non-detectable quantities ofmethanogens.

In various embodiments, the method further comprises identifying thesubject who has or is suspected to have a disease or condition caused byor associated with having a high methanogen quantity or a disease orcondition caused by or associated with having a low methanogen quantity,for treatment.

In various embodiments, the method further comprises obtaining orproviding the biological sample.

In various embodiments, the method further comprises subjecting thebiological sample to analysis for a quantity of a methanogen syntrophicmicroorganism. In various embodiments, the methanogen syntrophicmicroorganism is a hydrogen-producing microorganism. In variousembodiments, the method further comprises selecting a third therapy toinhibit the growth of the methanogen syntrophic microorganism. Invarious embodiments, the method further comprises administering thethird therapy. In various embodiments, the third therapy and the firsttherapy can be the same or same type of therapy.

Various embodiments of the present invention provide for a method fortreating a subject who has or is suspected to have a disease orcondition caused by or associated with having a high methanogen quantityor a disease or condition caused by or associated with having a lowmethanogen quantity. The method comprises administering a first therapyto the subject who has or is determined to have a methanogen quantitythat is higher than a reference value based on the recognition that thefirst therapy is appropriate for subjects who have a methanogen quantityhigher than the reference value, or administering a second therapy tothe subject who has or is determined to have a methanogen quantity thatis lower than the reference value based on the recognition that thesecond therapy is appropriate for subjects who have a methanogenquantity lower than the reference value.

First therapy and second therapy as used in this context do not refer toadministering two therapies, it is simply to provide a distinctionsbetween two types of therapies. The first therapy is a therapy that isappropriate for treating subjects who have high quantities ofmethanogens. The second therapy is a therapy that is appropriate fortreatment subject who have low or non-detectable quantities ofmethanogens.

In various embodiments, the method further comprises identifying thesubject who has or is suspected to have a disease or condition caused byor associated with having a high methanogen quantity or a disease orcondition caused by or associated with having a low methanogen quantity,for treatment.

In various embodiments, the subject has or is determined to have a highquantity of a methanogen syntrophic microorganism. In variousembodiments, the methanogen syntrophic microorganism is ahydrogen-producing microorganism. In various embodiments, the methodfurther comprises selecting a third therapy to inhibit the growth of themethanogen syntrophic microorganism. In various embodiments, the methodfurther comprises administering the third therapy. In variousembodiments, the third therapy and the first therapy can be the same orsame type of therapy.

Various embodiments of the present invention provide for a method ofdiagnosing disease or condition caused by or associated with having ahigh methanogen quantity or a disease or condition caused by orassociated with having a low methanogen quantity. The method comprisessubjecting a biological sample from a subject to analysis for methanogenquantity; comparing the methanogen quantity to a reference value; anddetermining that the subject has the disease or condition if themethanogen quantity is higher than the reference value, or determiningthat the subject does not have the disease or condition if themethanogen quantity is lower than the reference value.

In various embodiments, the method further comprises identifying thesubject for diagnosis. In various embodiments, the method furthercomprises obtaining or providing the biological sample.

In various embodiments, the method further comprises subjecting thebiological sample to analysis for a quantity of a methanogen syntrophicmicroorganism. In various embodiments, the methanogen syntrophicmicroorganism is a hydrogen-producing microorganism.

Various embodiments of the present invention provide for a method ofdiagnosing a susceptibility to a disease or condition caused by orassociated with having a high methanogen quantity. The method comprisessubjecting a biological sample from a subject to analysis for methanogenquantity; comparing the methanogen quantity to a reference value; anddetermining that the subject is susceptible to the disease or conditioncaused by or associated with having a high methanogen quantity if themethanogen quantity is higher than the reference value, or determiningthat the subject is not susceptible to the disease or condition causedby or associated with having a high methanogen quantity if themethanogen quantity is lower than the reference value.

Various embodiments of the present invention provide for a method ofdiagnosing a susceptibility to a disease or condition caused by orassociated with having a low methanogen quantity. The method comprisessubjecting a biological sample from a subject to analysis for methanogenquantity; comparing the methanogen quantity to a reference value; anddetermining that the subject is susceptible to the disease or conditioncaused by or associated with having a low methanogen quantity if themethanogen quantity is lower than the reference value, or determiningthat the subject is not susceptible to the disease or condition causedby or associated with having a low methanogen quantity if the methanogenquantity is higher than the reference value.

In various embodiments, the method further comprises identifying thesubject for diagnosis. In various embodiments, the method furthercomprises obtaining or providing the biological sample.

In various embodiments, the method further comprises subjecting thebiological sample to analysis for a quantity of a methanogen syntrophicmicroorganism. In various embodiments, the methanogen syntrophicmicroorganism is a hydrogen-producing microorganism.

In various embodiments, the invention provide for systems comprisingcomponents that are adapted to perform the methods of the inventiondescribed herein.

Reference Value

In various embodiments, the reference value is about 10,000 per ml ofthe biological sample. Thus, high methanogen quantity is a quantitygreater than 10,000 per ml of the biological sample, and a lowmethanogen quantity is a quantity less than 10,000 per ml of thebiological sample. In some embodiments, the reference value is about5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000,14,000, 15,000, or 20,000 per ml of the biological sample. Thus, in someembodiments, high methanogen quantity is a quantity greater than about5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000,14,000, 15,000, or 20,000 per ml of the biological sample, and a lowmethanogen quantity is a quantity less than 5,000, 6,000, 7,000, 8,000,9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, or 20,000 per mlof the biological sample. In some embodiments, these amounts can be permg of the biological sample.

In various embodiments, the reference value, particularly when used todetermine a low methanogen quantity is about 4,000 per ml of thebiological sample. Thus, low methanogen quantity is a quantity lowerthan 4,000 per ml of the biological sample. In some embodiments, thereference value is about 3,000, 2,000, 1,000, or 500 per ml of thebiological sample. Thus, in some embodiments, low methanogen quantity isa quantity less than 3,000, 2,000, 1,000, or 500 per ml of thebiological sample. In some embodiments, these amounts can be per mg ofthe biological sample.

The reference value can depend on the type of disease or condition thatwill be determined. Different types of diseases and conditions may havea different reference values.

In reference value can established from biological samples from ahealthy subject.

For example, if the biological sample is stool, then the reference valuecan be obtained from the stools of a healthy subject. In otherembodiments, the reference value is the average methanogen count for thesame type of biological sample from a population of healthy subjects. Inother embodiments, the reference value is the average plus one or twostandard deviations of average methanogen count for the same type ofbiological sample from a population of healthy subjects. In someembodiments, the population of healthy subjects can range from at leastthree healthy individuals to 25 healthy individuals, and even more than50 healthy individuals.

Subjects

The subject from whom a biological sample is obtained can be a subjectwho has or is suspected to have a disease or condition caused, at leastin part, by having high methanogen quantities or associated with havinghigh methanogen quantities. Examples of these subjects include but arenot limited to those who are or who are suspected to be overweight,obese, constipated, pre-diabetic, diabetic, insulin resistant, orglucose intolerant, or to have fatty liver (NASH).

The subjects from whom a biological sample is obtained can be a subjectwho has or is suspected to have a disease or condition caused, at leastin part, by having low methanogen quantities or associated with havinglow methanogen quantities. Examples of these subjects include but arenot limited to subjects who have or are suspected to have Crohn'sdisease or ulcerative colitis.

In certain embodiments the subject from whom a biological sample isobtained can be a subject who desires to know whether he or she issusceptible to a disease or condition caused, at least in part, byhaving high methanogen quantities or associated with having highmethanogen quantities. Examples of these subjects include but are notlimited to those who desires to know whether he or she are susceptibleto being overweight, obese, constipated, pre-diabetic, diabetic, insulinresistant, or glucose intolerant, or to have fatty liver (NASH).

The subjects from whom a biological sample is obtained can be a subjectwho desires to know whether he or she is susceptible to a disease orcondition caused, at least in part, by having low methanogen quantitiesor associated with having low methanogen quantities. Examples of thesesubjects include but are not limited to subjects who desires to knowwhether he or she is susceptible to having Crohn's disease or ulcerativecolitis.

Biological Samples

The biological sample that is analyzed by methods of the presentinvention can be stool, mucosal biopsy from a site in thegastrointestinal tract, or aspirated liquid from a site in thegastrointestinal tract. In various embodiments, the site in thegastrointestinal tract is mouth, stomach, small intestine, largeintestine, or anus. In various embodiments, the site in thegastrointestinal tract is duodenum, jejunum, or ileum. In variousembodiments, the site in the gastrointestinal tract is cecum, colon,rectum, or anus. In various embodiments, the site in thegastrointestinal tract is ascending colon, transverse colon, descendingcolon, or sigmoid flexure.

Diseases or Conditions

In various embodiments, the disease or condition caused, at least inpart, by having high methanogen quantities or associated with havinghigh methanogen quantities include but are not limited obesity,constipation, fatty liver (NASH), pre-diabetes, diabetes, insulinresistance, glucose and intolerance.

In various embodiments, the disease or condition caused, at least inpart, by having low methanogen quantities or associated with having lowmethanogen quantities include but are not limited Crohn's disease orulcerative colitis.

Therapies for Selection or Treatment of a Disease or Condition whenMethanogen Quantity is High

Therapies that directly inhibit the growth of methanogens and therebytreat disease or conditions caused by or related to high methanogenquantity, or reduce the likelihood of having disease or conditionscaused by or related to high methanogen quantity.

In various embodiments, once a high methanogen quantity is detected,these therapies can be administered alone or concurrently with a knowntherapy that treats the disease or condition as described herein.

In various embodiments an antibiotic or a combination of two or moreantibiotics can be selected and/or administered to subjects who havemethanogen quantity higher than the reference value. Examples ofantibiotics include but are not limited to aminoglycosides (e.g.,amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin,tobramycin, paromomycin), ansamycins (e.g., geldanamycin, herbimycin),carbacephems (e.g., loracarbef), carbapenems (e.g., ertapenem,doripenem, imipenem, cilastatin, meropenem), cephalosporins (e.g., firstgeneration: cefadroxil, cefazolin, cefalotin or cefalothin, cefalexin;second generation: cefaclor, cefamandole, cefoxitin, cefprozil,cefuroxime; third generation: cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone; fourth generation: cefepime; fifth generation:ceftobiprole), glycopeptides (e.g., teicoplanin, vancomycin), macrolides(e.g., azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, telithromycin, spectinomycin),monobactams (e.g., aztreonam), penicillins (e.g., amoxicillin,ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin,flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin,penicillin, piperacillin, ticarcillin), antibiotic polypeptides (e.g.,bacitracin, colistin, polymyxin b), quinolones (e.g., ciprofloxacin,enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin,norfloxacin, ofloxacin, trovafloxacin), rifamycins (e.g., rifampicin orrifampin, rifabutin, rifapentine, rifaximin), sulfonamides (e.g.,mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilamide,sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole, “tmp-smx”), andtetracyclines (e.g., demeclocycline, doxycycline, minocycline,oxytetracycline, tetracycline) as well as arsphenamine, chloramphenicol,clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid,furazolidone, isoniazid, linezolid, metronidazole, mupirocin,nitrofurantoin, platensimycin, pyrazinamide, quinupristin/dalfopristincombination, and tinidazole.

In various embodiments, the antibiotic selected and/or administered isrifaximin. The rifaximin therapy selected and/or administered can be200-2400 mg/dose, administered two or three times per day. In variousembodiments the dosage can be about 50, 75, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 mg/dose, In variousembodiments, the rifaximin therapy can be administered one, two, three,four or five times a day. In various embodiments, the therapy can beadministered for 5, 7, 10, 14, 15, 20, 21, or 28 days. In variousembodiments, the therapy can be re-administered after a period of notherapy.

In various embodiments, the antibiotic selected and/or administered isneomycin. The neomycin therapy selected and/or administered can be500-1000 mg/dose, administered two times per day. In various embodimentsthe dosage can be about 100, 200, 300, 400, 500, 600, 700, 750, 1000,1100, 1200, 1300, 1400, or 1500 mg/dose. In various embodiments, theneomycin therapy can be administered one, two, three, four or five timesa day. In various embodiments, the therapy can be administered for 5, 7,10, 14, 15, 20, 21, or 28 days. In various embodiments, the therapy canbe re-administered after a period of no therapy.

In various embodiments, the antibiotic selected and/or administered isvancomycin. The vancomycin therapy selected and/or administered can beabout 125 mg/dose, administered four times per day. In variousembodiments the dosage can be about 50, 75, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, or 500 mg/dose, In various embodiments, thevancomycin can be administered one, two, three, four or five times aday. In various embodiments, the therapy can be administered for 5, 7,10, 14, 15, 20, 21, or 28 days. In various embodiments, the therapy canbe re-administered after a period of no therapy.

In various embodiments, the antibiotic selected and/or administered ismetronidazole. The metronidazole therapy selected and/or administeredcan be 250-500 mg/dose, administered three times per day. In variousembodiments the dosage can be about 50, 75, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, or 1000mg/dose, In various embodiments, the metronidazole therapy can beadministered one, two, three, four or five times a day. In variousembodiments, the therapy can be administered for 5, 7, 10, 14, 15, 20,21, or 28 days. In various embodiments, the therapy can bere-administered after a period of no therapy.

In various embodiments, the two or more antibiotics selected and/oradministered are rifaximin and neomycin. In various embodiments, the twoor more antibiotics selected and/or administered are rifaximin andmetronidazole.

Particularly effective antibiotics may be non-absorbable antibiotics.Examples of non-absorbable antibiotics include but are not limited torifaximin, neomycin, Bacitracin, vancomycin, teicoplanin, ramoplanin,and paramomycin.

In some embodiments, a probiotic agent that inhibits the growth ofmethanogens, for example, Bifidobacterium sp. or Lactobacillus speciesor strains, e.g., L. acidophilus, L. rhamnosus, L. plantarum, L.reuteri, L. paracasei subsp. paracasei, or L. casei Shirota, orprobiotic Saccharomyces species, e.g., S. cerevisiae, is selected and/oradministered. The probiotic agent that inhibits the growth ofmethanogensis by typically administered in a pharmaceutically acceptableingestible formulation, such as in a capsule, or for some subjects,consuming a food supplemented with the inoculum is effective, forexample a milk, yogurt, cheese, meat or other fermentable foodpreparation. These probiotic agents can inhibit the growth ofmethanogens, for example, by competing against methanogens for growthand thus reduce or inhibit the growth of methanogens.

In various embodiments, the therapy selected and/or administered can bea reduced-calorie diet. This can be particularly beneficial for subjectswho are or are susceptible to being obese, or subjects who are or aresusceptible to being pre-diabetic, diabetic, insulin resistant, and/orglucose intolerant.

In various embodiments, the therapy selected and/or administered can bea reduced-fat diet. The inventors' research suggests that methanogengrowth increases in the presence of fat. Thus, a reduced-fat diet caninhibit the growth of methanogens and treat the diseases or conditionscaused by or related to high methanogen levels, or reduce the likelihoodof having these diseases or conditions.

In various embodiments, the therapy selected and/or administered can bean elemental diet. A comestible total enteral nutrition (TEN)formulation, which is also called an “elemental diet” are commerciallyavailable, for example, Vivonex® T.E.N. (Sandoz Nutrition, Minneapolis,Minn.) and its variants, or the like. A useful total enteral nutritionformulation satisfies all the subject's nutritional requirements,containing free amino acids, carbohydrates, lipids, and all essentialvitamins and minerals, but in a form that is readily absorbable in theupper gastrointestinal tract, thus depriving or “starving” themethanogen of nutrients of at least some of the nutrients theypreviously used for proliferating. Thus, methanogen growth is inhibited.

In various embodiments, the therapy selected and/or administered can bea selective inhibitor of methanogenesis, such as monensin or a statin(HMG-CoA reductase inhibitor). Statins can selectively inhibit thegrowth of methanogens without significantly inhibiting the growth ofnon-methanogens. Examples of statins include but are not limited toatorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin,rosuvastatin, and simvastatin.

Therapies That Can be Selected and/or Administered that Treats Diseaseor Conditions Caused by or Related to High Methanogen Level

In various embodiments, once a high methanogen quantity is detected,these therapies that treat the disease or condition can be administeredalone or along with a therapy that directly inhibits the growth ofmethanogens, as described herein.

Dosages and treatment regimens can be as indicated by the manufacturerfor the indicated disease or condition.

In various embodiments, the therapy selected and/or administered can bean anti-obesity drug. Examples of anti-obesity drugs include but are notlimited to phentermine, phentermine/topiramate, xenical, lorcaserin, andrimonabant.

In various embodiments, the therapy selected and/or administered can bea drug or a combination drug to treat pre-diabetes, diabetes, insulinresistance, or glucose intolerance. Examples of drugs include but arenot limited to alpha-glucosidase inhibitors, amylin analogs, dipeptidylpeptidase-4 inhibitors, GLP1 agonists, meglitinides, sulfonylureas,biguanides, thiazolidinediones (TZD), and insulin. Additional examplesof drugs include bromocriptine and welchol. Examples ofalpha-glucosidase inhibitors include but are not limited to acarbose andmiglitol. An example of an amylin analog is pramlintide. Examples ofdipeptidyl peptidase-4 inhibitors include but are not limited toSaxagliptin, Sitagliptin, Vildagliptin, Linagliptin, and Alogliptin.Examples of GLP1 agonist include but are not limited to liraglutide,exenatide, exenatide extended release. Examples of meglitinide includebut are not limited to nateglinide, and repaglinide. Examples ofsulfonylurea include but are not limited to chlorpropamide, Glimepiride,Glipizide, Glyburide, Tolazamide, and Tolbutamide. Examples of biguanideinclude but are not limited to Metformin, Riomet, Glucophage, GlucophageXR, Glumetza. Examples of thiazolidinedione include but are not limitedto Rosiglitazone and Pioglitazone. Examples of insulin include but arenot limited to Aspart, Detemir, Glargine, Glulisine, and Lispro.Examples of combination drugs include but are not limited toGlipizide/Metformin, Glyburide/Metformin, Pioglitazone/Glimepiride,Pioglitazone/Metformin, Repaglinide/Metformin,Rosiglitazone/Glimepiride, Rosiglitazone/Metformin,Saxagliptin/Metformin, Sitagliptin/Simvastatin, Sitagliptin/Metformin,Linagliptin/Metformin, Alogliptin/Metformin, andAlogliptin/Pioglitazone.

In various embodiments, the therapy selected and/or administered is totreat constipation. Examples of such therapies include, but are notlimited to laxatives, diet, guanylate cyclase C agonists, and serotoninagonists. An example of guanylate cyclase C agonist is linaclotide.Examples of serotonin agonists include prucalorpride and tegaserod.

In various embodiments, the therapy selected and/or administered is totreat fatty liver. An example of such therapy is metformin.

Therapies that can be Selected and/or Administered to Directly orIndirectly Treat Diseases and Conditions Caused by or Related to LowMethanogen Quantity or no Detectable Methanogens

In various embodiments, once a low methanogen quantity is detected or nodetectable methanogens is determined, these therapies that directlypromote the growth of methanogens or directly provides for colonizationof methanogens, can be administered alone or concurrently with knowntherapies that treat these diseases or condition.

In various embodiments, the therapy selected and/or administered is totreat Crohn's disease and/or ulcerative colitis. An example of suchtherapy is administering a methanogen. In various embodiments, themethanogen is from the genus Methanobrevibacter. In Examples ofMethanobrevibacter include but are not limited to M. acididurans, M.arboriphilus, M. curvatus, M. cuticularis, M. filiformis, M.gottschalkii, M. millerae, M. olleyae, M. oralis, M. ruminantium, M.smithii, M. thaueri, M. woesei, and M. wolinii. In certain embodiments,the Methanobrevibacter is Methanobrevibacter smithii (M. Smithii).

Pharmaceutical Compositions and Routes of Administration

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of a therapeutic agent describedherein.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

In certain embodiments, the therapeutic agents of the present inventionmay contain one or more acidic functional groups and, thus, are capableof forming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts, esters,amides, and prodrugs” as used herein refers to those carboxylate salts,amino acid addition salts, esters, amides, and prodrugs of the compoundsof the present invention which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use of the compounds of the invention. The term “salts”refers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds orby separately reacting the purified compound in its free base form witha suitable organic or inorganic acid and isolating the salt thus formed.These may include cations based on the alkali and alkaline earth metalssuch as sodium, lithium, potassium, calcium, magnesium and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cationsincluding, but not limited to ammonium, tetramethylanunonium, tetraethylammonium, methyl amine, dimethyl amine, trimethylamine, triethylamine,ethylamine, and the like (see, e.g., Berge S. M., et al. (1977) J.Pharm. Sci. 66, 1, which is incorporated herein by reference).

The term “pharmaceutically acceptable esters” refers to the relativelynontoxic, esterified products of the compounds of the present invention.These esters can be prepared in situ during the final isolation andpurification of the compounds, or by separately reacting the purifiedcompound in its free acid form or hydroxyl with a suitable esterifyingagent. Carboxylic acids can be converted into esters via treatment withan alcohol in the presence of a catalyst. The term is further intendedto include lower hydrocarbon groups capable of being solvated underphysiological conditions, e.g., alkyl esters, methyl, ethyl and propylesters.

As used herein, “pharmaceutically acceptable salts or prodrugs” aresalts or prodrugs that are, within the scope of sound medical judgment,suitable for use in contact with the tissues of subject without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use.

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the functionally active one or more peptides or drugs asdisclosed herein or a mutant, variant, analog or derivative thereof. Athorough discussion is provided in T. Higachi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A. C. S. Symposium Series,and in Bioreversible Carriers in: Drug Design, ed. Edward B. Roche,American Pharmaceutical Association and Pergamon Press, 1987, both ofwhich are hereby incorporated by reference. As used herein, a prodrug isa compound that, upon in vivo administration, is metabolized orotherwise converted to the biologically, pharmaceutically ortherapeutically active form of the compound. A prodrug of the one ormore peptides as disclosed herein or a mutant, variant, analog orderivative thereof can be designed to alter the metabolic stability orthe transport characteristics of one or more peptides or drugs asdisclosed herein or a mutant, variant, analog or derivative thereof, tomask side effects or toxicity, to improve the flavor of a compound or toalter other characteristics or properties of a compound. By virtue ofknowledge of pharmacodynamic processes and drug metabolism in vivo, oncea pharmaceutically active form of the one or more peptides or drugs asdisclosed herein or a mutant, variant, analog or derivative thereof,those of skill in the pharmaceutical art generally can design prodrugsof the compound (see, e.g., Nogrady (1985) Medicinal Chemistry ABiochemical Approach, Oxford University Press, N. Y., pages 388-392).Conventional procedures for the selection and preparation of suitableprodrugs are described, for example, in “Design of Prodrugs,” ed. H.Bundgaard, Elsevier, 1985. Suitable examples of prodrugs include methyl,ethyl and glycerol esters of the corresponding acid.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal or parenteral.

“Transdermal” administration may be accomplished using a topical creamor ointment or by means of a transdermal patch.

“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders. Compositions for parenteraluse may be provided in unit dosage forms (e.g., in single-doseampoules), or in vials containing several doses and in which a suitablepreservative may be added (see below).

The composition may be in form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in a form suitable for sterile injection. To preparesuch a composition, the suitable active agent(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, dextrose solution, and isotonic sodium chloride solution. Theaqueous formulation may also contain one or more preservatives (e.g.,methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of thecompounds is only sparingly or slightly soluble in water, a dissolutionenhancing or solubilizing agent can be added, or the solvent may include10-60% w/w of propylene glycol or the like.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Via the enteral route, the pharmaceutical compositions can be in theform of tablets, gel capsules, sugar-coated tablets, syrups,suspensions, solutions, powders, granules, emulsions, microspheres ornanospheres or lipid vesicles or polymer vesicles allowing controlledrelease. Via the parenteral route, the compositions may be in the formof solutions or suspensions for infusion or for injection.

Via the topical route, the pharmaceutical compositions based oncompounds according to the invention may be formulated for treating theskin and mucous membranes and are in the form of ointments, creams,milks, salves, powders, impregnated pads, solutions, gels, sprays,lotions or suspensions. They can also be in the form of microspheres ornanospheres or lipid vesicles or polymer vesicles or polymer patches andhydrogels allowing controlled release. These topical-route compositionscan be either in anhydrous form or in aqueous form depending on theclinical indication.

Via the ocular route, they may be in the form of eye drops.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Typical dosages of a therapeutically effective amount of the therapeuticagent can be in the ranges recommended by the manufacturer where knowntherapeutic compounds are used, and also as indicated to the skilledartisan by the in vitro responses or responses in animal models. Suchdosages typically can be reduced by up to about one order of magnitudein concentration or amount without losing the relevant biologicalactivity. Thus, the actual dosage can depend upon the judgment of thephysician, the condition of the patient, and the effectiveness of thetherapeutic method based.

Measuring Methanogens or Methanogen Syntrophic Microorganisms

In various embodiments, amplification-based assays can be used tomeasure the methanogen quantity or the quantity of methanogen syntrophicmicroorganisms. In such amplification-based assays, the nucleic acidsequences act as a template in an amplification reaction (e.g.,Polymerase Chain Reaction (PCR). In a quantitative amplification, theamount of amplification product will be proportional to the amount oftemplate in the original sample. Comparison to appropriate controls,e.g. healthy samples, provides a measure of the methanogen quantity.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis, et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). Measurement of DNA copy numberat microsatellite loci using quantitative PCR analysis is described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleicacid sequence for the genes is sufficient to enable one of skill in theart to routinely select primers to amplify any portion of the gene.Fluorogenic quantitative PCR may also be used in the methods of theinvention. In fluorogenic quantitative PCR, quantitation is based onamount of fluorescence signals, e.g., TaqMan and sybr green.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren, et al. (1988) Science 241:1077, and Barringer et al. (1990)Gene 89:117), transcription amplification (Kwoh, et al. (1989) Proc.Natl. Acad. Sci. USA 86:1173), self-sustained sequence replication(Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87:1874), dot PCR,and linker adapter PCR, etc.

In still other embodiments of the methods provided herein, sequencing ofindividual nucleic molecules (or their amplification products) isperformed. In one embodiment, a high throughput parallel sequencingtechnique that isolates single nucleic acid molecules of a population ofnucleic acid molecules prior to sequencing may be used. Such strategiesmay use so-called “next generation sequencing systems” including,without limitation, sequencing machines and/or strategies well known inthe art, such as those developed by Illumina/Solexa (the GenomeAnalyzer; Bennett et al. (2005) Pharmacogenomics, 6:373-20 382), byApplied Biosystems, Inc. (the SOLiD Sequencer;solid.appliedbiosystems.com), by Roche (e.g., the 454 GS FLX sequencer;Margulies et al. (2005) Nature, 437:376-380; U.S. Pat. Nos. 6,274,320;6,258,568; 6,210,891), by Heliscope™ system from Helicos Biosciences(see, e.g., U.S. Patent App. Pub. No. 2007/0070349), and by others.Other sequencing strategies such as stochastic sequencing (e.g., asdeveloped by Oxford Nanopore) may also be used, e.g., as described inInternational Application No. PCT/GB2009/001690 (pub. no.WO/2010/004273).

In still other embodiments of the methods provided herein, deepsequencing can be used to identify and quantify the methanogen ormethanogen syntrophic microorganism. These techniques are known in theart.

Nucleic Acid Sample Preparation

A. Nucleic Acid Isolation

Nucleic acid samples derived from the biological sample from a subjectthat can be used in the methods of the invention to determine themethanogen quantity can be prepared by means well known in the art. Forexample, surgical procedures or needle biopsy aspiration can be used tobiological samples from a subject.

In one embodiment, the nucleic acid samples used to compute a referencevalue are taken from at least 1, 2, 5, 10, 20, 30, 40, 50, 100, or 200different organisms of that species. According to certain aspects of theinvention, nucleic acid “derived from” genomic DNA, as used in themethods of the invention can be fragments of genomic nucleic acidgenerated by restriction enzyme digestion and/or ligation to othernucleic acid, and/or amplification products of genomic nucleic acids, orpre-messenger RNA (pre-mRNA), amplification products of pre-mRNA, orgenomic DNA fragments grown up in cloning vectors generated, e.g., by“shotgun” cloning methods. In certain embodiments, genomic nucleic acidsamples are digested with restriction enzymes.

B. Amplification of Nucleic Acids

Though the nucleic acid sample need not comprise amplified nucleic acid,in some embodiments, the isolated nucleic acids can be processed inmanners requiring and/or taking advantage of amplification. The genomicDNA samples of the biological sample from a subject optionally can befragmented using restriction endonucleases and/or amplified prior todetermining analysis. In one embodiment, the DNA fragments are amplifiedusing polymerase chain reaction (PCR). Methods for practicing PCR arewell known to those of skill in the art. One advantage of PCR is thatsmall quantities of DNA can be used. For example, genomic DNA from thebiological sample from a subject may be about 150 ng, 175, ng, 200 ng,225 ng, 250 ng, 275 ng, or 300 ng of DNA.

In certain embodiments of the methods of the invention, the nucleic acidfrom a biological sample of a subject is amplified using a single primerpair. For example, genomic DNA samples can be digested with restrictionendonucleases to generate fragments of genomic DNA that are then ligatedto an adaptor DNA sequence which the primer pair recognizes. In otherembodiments of the methods of the invention, the nucleic acid from abiological sample of a subject is amplified using sets of primer pairsspecific to methanogens. Such sets of primer pairs each recognizegenomic DNA sequences flanking the gene wherein the methanogen detectionor quantification is also to be assessed. A DNA sample suitable forhybridization can be obtained, e.g., by polymerase chain reaction (PCR)amplification of genomic DNA, fragments of genomic DNA, fragments ofgenomic DNA ligated to adaptor sequences or cloned sequences. Computerprograms that are well known in the art can be used in the design ofprimers with the desired specificity and optimal amplificationproperties, such as Oligo version 5.0 (National Biosciences). PCRmethods are well known in the art, and are described, for example, inInnis et al., eds., 1990, PCR Protocols: A Guide to Methods AndApplications, Academic Press Inc., San Diego, Calif. It will be apparentto one skilled in the art that controlled robotic systems are useful forisolating and amplifying nucleic acids and can be used.

In other embodiments, where genomic DNA of a biological sample from asubject is fragmented using restriction endonucleases and amplifiedprior to analysis, the amplification can comprise cloning regions ofgenomic DNA of a biological sample from a subject. In such methods,amplification of the DNA regions is achieved through the cloningprocess. For example, expression vectors can be engineered to expresslarge quantities of particular fragments of genomic DNA of a biologicalsample from a subject (Sambrook, J. et al., eds., 1989, MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., at pp. 9.47-9.51).

In yet other embodiments, where the DNA of a biological sample from asubject is fragmented using restriction endonucleases and amplifiedprior to analysis, the amplification comprises expressing a nucleic acidencoding a gene, or a gene and flanking genomic regions of nucleicacids, from the biological sample from the subject. RNA (pre-messengerRNA) that comprises the entire transcript including introns is thenisolated and used in the methods of the invention to analyze and providea methanogen quantity. In certain embodiments, no amplification isrequired. In such embodiments, the genomic DNA, or pre-RNA, from the abiological sample of a subject may be fragmented using restrictionendonucleases or other methods. The resulting fragments may behybridized to SNP probes. Typically, greater quantities of DNA areneeded to be isolated in comparison to the quantity of DNA or pre-mRNAneeded where fragments are amplified. For example, where the nucleicacid from a biological sample of a subject is not amplified, a DNAsample from a biological sample of a subject for use in hybridizationmay be about 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ngof DNA or greater. Alternatively, in other embodiments, methods are usedthat require very small amounts of nucleic acids for analysis, such asless than 400 ng, 300 ng, 200 ng, 100 ng, 90 ng, 85 ng, 80 ng, 75 ng, 70ng, 65 ng, 60 ng, 55 ng, 50 ng, or less, such as is used for molecularinversion probe (MIP) assays. These techniques are particularly usefulfor analyzing clinical samples, such as paraffin embedded formalin-fixedmaterial or small core needle biopsies, characterized as being readilyavailable but generally having reduced DNA quality (e.g., small,fragmented DNA) and/or not providing large amounts of nucleic acids.

C. Hybridization

The nucleic acid samples derived from a biological sample from a subjectused in the methods of the invention can be hybridized to arrayscomprising probes (e.g., oligonucleotide probes) in order to identifyand/or quantify methanogens. In certain embodiments, the probes used inthe methods of the invention comprise an array of probes that can betiled on a DNA chip (e.g., SNP oligonucleotide probes). In someembodiments, the methanogen is determined by a method that does notcomprise detecting a change in size of restriction enzyme-digestednucleic acid fragments. In other embodiments, SNPs are analyzed toidentify or quantify the methanogen. Hybridization and wash conditionsused in the methods of the invention are chosen so that the nucleic acidsamples to be analyzed by the invention specifically bind orspecifically hybridize to the complementary oligonucleotide sequences ofthe array, preferably to a specific array site, wherein itscomplementary DNA is located. In some embodiments, the complementary DNAcan be completely matched or mismatched to some degree as used, forexample, in Affymetrix oligonucleotide arrays such as those used toanalyze SNPs in MIP assays. The single-stranded syntheticoligodeoxyribonucleic acid DNA probes of an array may need to bedenatured prior to contact with the nucleic acid samples of a biologicalsample from a subject, e.g., to remove hairpins or dimers which form dueto self-complementary sequences.

Optimal hybridization conditions will depend on the length of the probesand type of nucleic acid samples from a biological sample from asubject. General parameters for specific (i.e., stringent) hybridizationconditions for nucleic acids are described in Sambrook, J. et al., eds.,1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., at pp. 9.47-9.51 and11.55-11.61; Ausubel et al., eds., 1989, Current Protocols in MoleculesBiology, Vol. 1, Green Publishing Associates, Inc., John Wiley & Sons,Inc., New York, at pp. 2.10.1-2.10.16. Exemplary useful hybridizationconditions are provided in, e.g., Tijessen, 1993, Hybridization withNucleic Acid Probes, Elsevier Science Publishers B. V. and Kricka, 1992,Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Calif.

D. Oligonucleotide Nucleic Acid Arrays

In some embodiments of the methods of the present invention, DNA arrayscan be used to assess methanogen quantity that comprise complementarysequences. Hybridization can be used to determine the presence and/orquantity of methanogens. Various formats of DNA arrays that employoligonucleotide “probes,” (i.e., nucleic acid molecules having definedsequences) are well known to those of skill in the art. Typically, a setof nucleic acid probes, each of which has a defined sequence, isimmobilized on a solid support in such a manner that each differentprobe is immobilized to a predetermined region. In certain embodiments,the set of probes forms an array of positionally-addressable binding(e.g., hybridization) sites on a support. Each of such binding sitescomprises a plurality of oligonucleotide molecules of a probe bound tothe predetermined region on the support. More specifically, each probeof the array is preferably located at a known, predetermined position onthe solid support such that the identity (i.e., the sequence) of eachprobe can be determined from its position on the array (i.e., on thesupport or surface). Microarrays can be made in a number of ways, ofwhich several are described herein. However produced, microarrays sharecertain characteristics, they are reproducible, allowing multiple copiesof a given array to be produced and easily compared with each other.

In certain embodiments, the microarrays are made from materials that arestable under binding (e.g., nucleic acid hybridization) conditions. Themicroarrays are preferably small, e.g., between about 1 cm² and 25 cm²,preferably about 1 to 3 cm². However, both larger and smaller arrays arealso contemplated and may be preferable, e.g., for simultaneouslyevaluating a very large number of different probes. Oligonucleotideprobes can be synthesized directly on a support to form the array. Theprobes can be attached to a solid support or surface, which may be made,e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide,nitrocellulose, gel, or other porous or nonporous material. The set ofimmobilized probes or the array of immobilized probes is contacted witha sample containing labeled nucleic acid species so that nucleic acidshaving sequences complementary to an immobilized probe hybridize or bindto the probe. After separation of, e.g., by washing off, any unboundmaterial, the bound, labeled sequences are detected and measured. Themeasurement is typically conducted with computer assistance. Using DNAarray assays, complex mixtures of labeled nucleic acids, e.g., nucleicacid fragments derived a restriction digestion of genomic DNA, can beanalyzed.

In certain embodiments, high-density oligonucleotide arrays are used inthe methods of the invention. These arrays containing thousands ofoligonucleotides complementary to defined sequences, at definedlocations on a surface can be synthesized in situ on the surface by, forexample, photolithographic techniques (see, e.g., Fodor et al., 1991,Science 251:767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A.91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; U.S.Pat. Nos. 5,578,832; 5,556,752; 5,510,270; 5,445,934; 5,744,305; and6,040,138). Methods for generating arrays using inkjet technology for insitu oligonucleotide synthesis are also known in the art (see, e.g.,Blanchard, International Patent Publication WO 98/41531, published Sep.24, 1998; Blanchard et al., 1996, Biosensors And Bioelectronics11:687-690; Blanchard, 1998, in Synthetic DNA Arrays in GeneticEngineering, Vol. 20, J. K. Setlow, Ed., Plenum Press, New York at pages111-123). Another method for attaching the nucleic acids to a surface isby printing on glass plates, as is described generally by Schena et al.(1995, Science 270:467-470). Other methods for making microarrays, e.g.,by masking (Maskos and Southern, 1992, Nucl. Acids. Res. 20:1679-1684),may also be used. When these methods are used, oligonucleotides (e.g.,15 to 60-mers) of known sequence are synthesized directly on a surfacesuch as a derivatized glass slide. The array produced can be redundant,with several oligonucleotide molecules.

One exemplary means for generating the oligonucleotide probes of the DNAarray is by synthesis of synthetic polynucleotides or oligonucleotides,e.g., using N-phosphonate or phosphoramidite chemistries (Froehler etal., 1986, Nucleic Acid Res. 14:5399-5407; McBride et al., 1983,Tetrahedron Lett. 24:246-248). Synthetic sequences are typically betweenabout 15 and about 600 bases in length, more typically between about 20and about 100 bases, most preferably between about 40 and about 70 basesin length. In some embodiments, synthetic nucleic acids includenon-natural bases, such as, but by no means limited to, inosine. Asnoted above, nucleic acid analogues may be used as binding sites forhybridization. An example of a suitable nucleic acid analogue is peptidenucleic acid (see, e.g., Egholm et al., 1993, Nature 363:566-568; U.S.Pat. No. 5,539,083). In alternative embodiments, the hybridization sites(i.e., the probes) are made from plasmid or phage clones of regions ofgenomic DNA corresponding to SNPs or the complement thereof. The size ofthe oligonucleotide probes used in the methods of the invention can beat least 10, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. It iswell known in the art that although hybridization is selective forcomplementary sequences, other sequences which are not perfectlycomplementary may also hybridize to a given probe at some level. Thus,multiple oligonucleotide probes with slight variations can be used, tooptimize hybridization of samples. To further optimize hybridization,hybridization stringency condition, e.g., the hybridization temperatureand the salt concentrations, may be altered by methods that are wellknown in the art.

In certain embodiments, the high-density oligonucleotide arrays used inthe methods of the invention comprise oligonucleotides corresponding toa methanogen.

E. Labeling

In some embodiments, the nucleic acids samples, fragments thereof, orfragments thereof used in the methods of the invention are detectablylabeled. For example, the detectable label can be a fluorescent label,e.g., by incorporation of nucleotide analogues. Other labels suitablefor use in the present invention include, but are not limited to,biotin, iminobiotin, antigens, cofactors, dinitrophenol, lipoic acid,olefinic compounds, detectable polypeptides, electron rich molecules,enzymes capable of generating a detectable signal by action upon asubstrate, and radioactive isotopes.

Radioactive isotopes include that can be used in conjunction with themethods of the invention, but are not limited to, 32P and 14C.Fluorescent molecules suitable for the present invention include, butare not limited to, fluorescein and its derivatives, rhodamine and itsderivatives, texas red, 5′carboxy-fluorescein (“FAM”), 2′,7′-dimethoxy-4′, 5′-dichloro-6-carboxy-fluorescein (“JOE”), N, N, N′,N′-tetramethyl-6-carboxy-rhodamine (“TAMRA”), 6-carboxy-X-rhodamine(“ROX”), HEX, TET, IRD40, and IRD41.

Fluorescent molecules which are suitable for use according to theinvention further include: cyanine dyes, including but not limited toCy2, Cy3, Cy3.5, CY5, Cy5.5, Cy7 and FLUORX; BODIPY dyes including butnot limited to BODIPY-FL, BODIPY-TR, BODIPY-TMR, BODIPY-630/650, andBODIPY-650/670; and ALEXA dyes, including but not limited to ALEXA-488,ALEXA-532, ALEXA-546, ALEXA-568, and ALEXA-594; as well as otherfluorescent dyes which will be known to those who are skilled in theart. Electron rich indicator molecules suitable for the presentinvention include, but are not limited to, ferritin, hemocyanin, andcolloidal gold.

Two-color fluorescence labeling and detection schemes may also be used(Shena et al., 1995, Science 270:467-470). Use of two or more labels canbe useful in detecting variations due to minor differences inexperimental conditions (e.g., hybridization conditions). In someembodiments of the invention, at least 5, 10, 20, or 100 dyes ofdifferent colors can be used for labeling. Such labeling would alsopermit analysis of multiple samples simultaneously which is encompassedby the invention.

The labeled nucleic acid samples, fragments thereof, or fragmentsthereof ligated to adaptor regions that can be used in the methods ofthe invention are contacted to a plurality of oligonucleotide probesunder conditions that allow sample nucleic acids having sequencescomplementary to the probes to hybridize thereto. Depending on the typeof label used, the hybridization signals can be detected using methodswell known to those of skill in the art including, but not limited to,X-Ray film, phosphor imager, or CCD camera. When fluorescently labeledprobes are used, the fluorescence emissions at each site of a transcriptarray can be, preferably, detected by scanning confocal lasermicroscopy. In one embodiment, a separate scan, using the appropriateexcitation line, is carried out for each of the two fluorophores used.Alternatively, a laser can be used that allows simultaneous specimenillumination at wavelengths specific to the two fluorophores andemissions from the two fluorophores can be analyzed simultaneously (seeShalon et al. (1996) Genome Res. 6, 639-645). In a preferred embodiment,the arrays are scanned with a laser fluorescence scanner with a computercontrolled X-Y stage and a microscope objective. Sequential excitationof the two fluorophores is achieved with a multi-line, mixed gas laser,and the emitted light is split by wavelength and detected with twophotomultiplier tubes. Such fluorescence laser scanning devices aredescribed, e.g., in Schena et al. (1996) Genome Res. 6, 639-645.Alternatively, a fiber-optic bundle can be used such as that describedby Ferguson et al. (1996) Nat. Biotech. 14, 1681-1684. The resultingsignals can then be analyzed to determine methanogen quantity, usingcomputer software.

F. Algorithms for Analyzing Methanogen Quantity

Once the hybridization signal has been detected the resulting data canbe analyzed using algorithms. In certain embodiments, the algorithm forquantitating methanogens is based on well-known methods.

G. Computer Implementation Systems and Methods

In certain embodiments, the methods of the invention implement acomputer program to calculate methanogen quantity. For example, acomputer program can be used to perform the algorithms described herein.A computer system can also store and manipulate data generated by themethods of the present invention which comprises a plurality ofhybridization signal changes/profiles during approach to equilibrium indifferent hybridization measurements and which can be used by a computersystem in implementing the methods of this invention. In certainembodiments, a computer system receives probe hybridization data; (ii)stores probe hybridization data; and (iii) compares probe hybridizationdata to determine the quantity of methanogen. Whether the quantity ishigher or lower than the reference value is calculated. In someembodiments, a computer system (i) compares the methanogen quantity to athreshold value or reference value; and (ii) outputs an indication ofwhether said methanogen quantity is above or below a threshold orreference value, or the presence of a disease or condition based on saidindication. In certain embodiments, such computer systems are alsoconsidered part of the present invention.

Numerous types of computer systems can be used to implement the analyticmethods of this invention according to knowledge possessed by a skilledartisan in the bioinformatics and/or computer arts.

Several software components can be loaded into memory during operationof such a computer system. The software components can comprise bothsoftware components that are standard in the art and components that arespecial to the present invention (e.g., dCHIP software described in Linet al. (2004) Bioinformatics 20, 1233-1240; CRLMM software described inSilver et al. (2007) Cell 128, 991-1002; Aroma Affymetrix softwaredescribed in Richardson et al. (2006) Cancer Cell 9, 121-132. Themethods of the invention can also be programmed or modeled inmathematical software packages that allow symbolic entry of equationsand high-level specification of processing, including specificalgorithms to be used, thereby freeing a user of the need toprocedurally program individual equations and algorithms. Such packagesinclude, e.g., Matlab from Mathworks (Natick, Mass.), Mathematica fromWolfram Research (Champaign, Ill.) or S-Plus from MathSoft (Seattle,Wash.). In certain embodiments, the computer comprises a database forstorage of hybridization signal profiles. Such stored profiles can beaccessed and used to calculate a methanogen quantity.

Kits

The present invention is also directed to a kit for the determination,selection, and/or treatment of the disease or conditions describedherein. The kit is an assemblage of materials or components, includingat least one of the inventive compositions. Thus, in some embodimentsthe kit contains a composition including a therapeutic agent, asdescribed above. In other embodiments, the kit contains primers for thequantification of methanogens or methanogen syntrophic microorganisms.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. In one embodiment, the kit isconfigured particularly for the purpose of treating mammalian subjects.In another embodiment, the kit is configured particularly for thepurpose of treating human subjects. In further embodiments, the kit isconfigured for veterinary applications, treating subjects such as, butnot limited to, farm animals, domestic animals, and laboratory animals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to quantify the methanogens or methanogen syntrophicmicroorganisms, or to inhibit the growth of methanogens or methanogensyntrophic microorganisms, or to treat disease or conditions caused byor associated with methanogens or methanogen syntrophic microorganismsOptionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, bandagingmaterials or other useful paraphernalia as will be readily recognized bythose of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.As used herein, the term “package” refers to a suitable solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding the individual kit components. The packaging material generallyhas an external label which indicates the contents and/or purpose of thekit and/or its components.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1

Patient Inclusion and Exclusion Criteria

The study was approved by the inventors' institutional review board, andinformed consent was obtained from all participants. Consecutive Rome IIpositive IBS subjects aged 18-65 years of age who presented forlactulose breath testing were eligible for the study. Patients wereexcluded if they had any of the following: a history of abdominalsurgery such as bowel resection (except cholecystectomy orappendectomy), known intestinal disorder such as inflammatory boweldisease, abdominal adhesions, perirectal or intestinal fistula, unstablethyroid disease, diabetes, cancer, HIV, pregnancy, use of medicationsknown to affect intestinal motility such as narcotics, imodium, andtegaserod, or antibiotic usage within the past 1 month.

Collection of Breath and Stool Samples

All patients were first asked to complete a bowel symptom questionnairein order to determine the relative degree of constipation to diarrheabased on C-D VAS scoring as previously validated [13]. Subjects thenunderwent lactulose breath testing (LBT). As part of the LBT, subjectswere asked to ingest 10 gm oral lactulose in solution (PharmaceuticalAssociates, Inc., Greenville, S.C.) after a baseline breath sample.Lactulose is a polysaccharide that is not digested by humans, but can beutilized by enteric flora. Repeat breath samples were then obtainedevery 15 minutes after lactulose ingestion until 180 minutes, and levelsof methane and hydrogen were analyzed using gas chromatography (Quintroninstrument company, Milwaukee, Wis.). A positive methane breath test wasdefined as a breath methane level≥3 ppm as previously published [5, 13].Using the questionnaire and breath test results, subjects who hadmethane on breath analysis and constipation predominant IBS wereselected. The control group included those with any form of IBS who didnot test positive for methane on breath testing. After completion ofbreath testing, all subjects were provided a stool container andinstructions on how to collect the stool sample. Patients returned thestool sample that was fresh frozen within 24 hours of collection.

Stool PCR Testing

From each stool sample, bacterial DNA was extracted using QIAamp PCR kit(Qiagen, Hilden, Germany). PCR (Eppendorf mastercycler gradient) withpreviously published universal 16S rDNA primer was used to detect thepresence of total bacteria in stool. Quantitative-PCR was performed onthe same stool samples using the rpoB gene primer specific for M.smithii only (Table 1). In addition, quantitative PCR was also conductedto determine total bacteria count using universal primers (Table 1).

TABLE 1 Various PCR primers used to detect bacterial DNA in stoolAmplicon SEQ ID Organism Target Primers (5'-3') Size NO Universal 16STCCTACGGGAGGCAGCAGT 466 bp 1 rDNA GGACTACCAGGGTATCTAATCCTGTT 2M. Smithii rpoB AAGGGATTTGCACCCAACAC  70 bp 3 GACCACAGTTAGGACCCTCTGG 4

Quantitative PCR was performed with the CFX96™ Real-Time PCR DetectionSystem (Bio-Rad Laboratories, Hercules, Calif.) using optical grade96-well plates. Duplicate samples were routinely used for thedetermination of DNA by real-time PCR. The PCR reaction was performed ina total volume of 20 μl using the iQ™ SYBR GREEN Supermix (Bio-Radlaboratories), containing 300 nM each of the universal forward andreverse primers. The reaction conditions were set at 95° C. for 3 minfollowed by 40 cycles at 95° C. for 10 s, 55° C. for 10 s and 72° C. for30 s then 95° C. for 10 s. Data analysis made use of CFX Managersoftware supplied by Bio-Rad. To generate standard curves for totalbacteria, the Ct values were plotted relative to the template DNAextracted from corresponding serial tenfold dilution of cultures ofEscherichia coli strain ATCC 25922. Escherichia coli strain ATCC 25922was previously grown in TB Growth Media (MO BIO Laboratories, Inc.Carlsbad, Calif.) to a concentration of 10⁸ CFU then plated on LB (ISCBioExpress, Kaysville, Utah) agar plates to verify colony counts. The10⁸ CFU Escherichia coli solution was subjected to DNA extraction byusing a Qiaamp® DNA Mini kit (Qiagen). The extracted DNA was used tocreate ten-fold dilutions and establish a standard curve. Similarly,calibration curves for M Smithii were made by aliquoting ten-folddilutions of 10⁸ CFU M. Smithii liquid culture. Concentration wasdetermined by measuring optical density at 600nm.

Statistical Analysis

Mann Whitney U test was utilized for non-parametric data and student'st-test was used for normally distributed data. The quantity of M.smithii was compared to the amount of methane on breath testing usingSpearman rank correlation. Comparing breath test parts per millionbetween hydrogen and methane utilized Pearson regression analysis. Inaddition, M. smithii was represented as a ratio percent to the combinedtotal bacteria and M. smithii counts and this percent was also comparedto breath test status, methane levels and degree of constipation. Alltests were two-tailed and statistical significance was defined asP<0.05.

Baseline Characteristics

A total of 9 patients (C-IBS with positive methane breath analysis) and10 controls (IBS with no breath methane) met the inclusion criteria. Themajority of subjects in each group were females (8 of 9 in methane groupand 8 of 10 among the non-methane controls). The average age was nodifferent between the two groups (43.8±8.7 years in methane positive vs.41.9±9.9 years in methane negative subjects). The validated symptom C-Dscore (range of score from −100 to +100) was 51.1±37.8 mm in the C-IBSwith methane group which was greater than −1.0±35.1 mm for non-methanesubjects (P<0.01) indicating significant constipation in methanepositive subjects relative to diarrhea. There was no difference inbloating or abdominal pain severity between the groups.

PCR Results from Stool

On q-PCR, M. smithii samples were not interpretable due to poor samplein 2 methane and 1 hydrogen producing subjects leaving 7 methane and 9non-methane producing subjects eligible for analysis. In the case ofq-PCR for total bacterial counts, 6 samples were not interpretableleaving 13 (6 breath methane positive and 7 breath methane negative) foranalysis. In determining the percent M. smithii, there were 12 samplesin which both M. smithii and bacterial levels were measured.

Examining M. smithii first, M. smithii was detected in both methaneproducers as well as non-methane subjects. However, the presence of M.smithii was significantly higher in breath methane positive subjects(1.8×10⁷±3.0×10⁷ copies per gm wet stool) as compared to those withnegative breath methane (3.2×10⁵±7.6×10⁵ copies per gm wet stool)(p<0.001). Based on these findings, the minimum threshold of M. smithiiin order to produce positive lactulose breath testing for methane wasdeemed to be 4.2×10⁵ copies per gm of wet stool (FIG. 1).

To further evaluate this relationship, the ratio of M. smithii to thecombined total bacteria and M. smithii was expressed as a percent. Inthe non-methane producers, the percent M. smithii was 0.24±0.47% andamong methane producing subjects was 7.1±6.3% (P=0.02) (FIG. 2). Basedon percent counts, M. smithii greater than 1.2% was always indicative ofpositive breath methane.

Comparing M. smithii and Breath Methane and Hydrogen Levels on BreathTest

The amount of breath methane produced as determined by 180 min AUCcorrelated significantly with the quantity of M. smithii in stool(R=0.76, P<0.001) (FIG. 3). While total bacterial counts did notcorrelate with methane on breath testing, the percent M. smithii washighly correlated with the level of methane on breath test (R=0.77,P=0.001) (FIG. 4).

In contrast to methane, when breath hydrogen was compared to quantitiesof M. smithii, total prokaryotic bacteria and the percent of M. smithii,no trend was seen. However, there was an expected hydrogen utilizationby methane as suggested by an inverse correlation between breath methaneAUC and hydrogen AUC (R=−0.61, P=0.005) (FIG. 5).

Constipation Symptoms, M. smithii and Total Bacterial Count

Using the previously validated score examining constipation as arelative value to diarrhea (C-D), the inventors examined if M. smithiiand total bacterial levels were predictive of constipation severity.Both absolute M. smithii (FIG. 6) (R=0.43, P=0.1) and percent M. smithii(FIG. 7) (R=0.47, P=0.12) did not quite meet significance in acomparison to the severity of constipation by C-D. Also in the case ofM. smithii and percent M. smithii, no correlation was seen betweenlevels and severity of abdominal pain or bloating.

In the case of total bacterial counts, there was no association with C-Dscore and no association with bloating. Although, there was an inversecorrelation between bacterial levels and abdominal pain VAS scores(R=−0.51), it did not reach statistical significance (P=0.07) (FIG. 8).

Example 2

M. smithii Hypercolonization of Rats

Twenty adult Sprague-Dawley rats were obtained as 21-day-old weanlings(Harlan Labs, Indianapolis, Ind.). After 3 days of quarantine, all ratswere weighed, and then received a 1 ml aliquot of 5% sodium bicarbonateby oral gavage using a ball-tipped inoculating needle, in order toneutralize the gastric acid. After ˜20 min, one group of rats (n=10)received a 0.5 ml gavage of M. smithii in liquid growth media. A secondgroup of rats (n=10) received a 0.5 ml gavage of liquid growth media.After another 20 min, rats gavaged with M. smithii were given 0.2 mlenemas of the same inoculate following isoflurane anesthesia in adessicator jar. The gavage and enemas were performed to determinewhether M. smithii levels in intestine could be enhanced throughhypercolonization.

Tracking Colonization by M. smithii

After inoculation, all rats were housed two per microisolater cage understandard vivarium procedures and maintained on normal rodent chow (5.7%fat) (Lab Rodent Diet 5001; Newco Distributors, Rancho Cucamonga,Calif.). Fresh stool samples were collected daily for the first week,and then approximately every 2 weeks thereafter.

The stool specimens on specific weeks were tested for the levels of M.smithii and of total bacteria by performing qPCR as previously described(27). M. smithii levels were quantitated using primers for the RpoB gene(5′-AAGGGATTTGCACCCAACAC-3′ (forward) (SEQ ID NO:3) and5′-GACCACAGTTAGGACCCTCTGG-3′ (reverse) (SEQ ID NO:4)) and total bacteriawere quantitated using 16S recombinant DNA (5′-TCCTACGGGAGGCAGCAGT-3′(forward) (SEQ ID NO:1) and 5′-GGACTACCAGGGTATCTAATCCTGTT-3′ (reverse)(SEQ ID NO:2)). Animal weights were also obtained once a week.

Diet Manipulation

Rats were observed until three consecutive weights were obtained within10 g to suggest an end of growth curve and arrival at adult weight(corresponded to day 112). On day 112, all rats were then switched to ahigh-fat diet (34.3% fat) (Teklad high fat diet TD.06414; HarlanLaboratories, Madison, Wis.) and maintained on this diet for 10 weeksuntil day 182. Fresh stool samples and animal weights were collectedfrom all animals on a weekly basis. On day 182, all rats were returnedto normal chow. Finally, on day 253, five rats from each group wereagain fed high-fat chow. The rats were maintained on their respectivediets while stool samples and weekly weights continued to be obtainedfor 5 weeks until euthanasia at day 287. This last phase was toguarantee that 10 rats were on high-fat and 10 on normal chow for aperiod of time before euthanasia.

Euthanasia and Bowel Sampling

On day 287 post-inoculation, all rats were euthanized by CO2asphyxiation and pneumothorax. Laparotomy was performed and sections ofthe left colon, cecum, ileum, jejunum, and duodenum were resected fromeach rat as previously described (A27). DNA was extracted from luminalcontents of each segment as previously described (A27), and qPCR with M.smithii-specific and universal bacterial primers was performed todetermine the levels of M. smithii and total bacteria, respectively ineach segment. The study protocol was approved by the Cedars-SinaiInstitutional Animal Care Utilization Committee (IACUC).

Statistical Analysis

The levels of M. smithii in stool by qPCR between inoculated andnoninoculated rats were compared by Mann-Whitney U-test. Comparisons ofbody weight before and after diet changes were compared by pairedt-test. Levels of M. smithii in bowel segments or stool between groupswere again compared by Mann-Whitney U-test. For comparison of M. smithiilevels before and after an intervention, Wilcoxon signed-rank test wasused. For weights, data were expressed as mean±s.d. and data for M.smithii levels were expressed as mean±s.e. Statistical significance wasdetermined by P<0.05.

Colonization of Rats with M. smithii

At baseline, all rats demonstrated the presence of M. smithii in thestool which were not different between groups (FIG. 9).

After inoculation with M. smithii, rats demonstrated an increaseddetection of stool M. smithii than control animals. However, this didnot persist as levels returned to control levels by day 9 (FIG. 9).Since hypercolonization did not occur, in the remaining experiments allof the rats were examined as a single group.

M. smithii Levels and Weight After Initial Transition to High-Fat Diet

All rats were initially fed normal rat chow until three consecutiveweights were obtained within a 10 g plateau to suggest the rats hadreached adult weight. This plateau occurred in the 2 weeks preceding day112 (FIG. 10a ). During three consecutive measurements obtained betweendays 98 and 112, there was only a mean change in weight of 5.5±5.8 g.After switching to high-fat chow on day 112, a sudden increase in ratweights was observed (FIG. 10a ). The average weight increased from268±13 g on day 112 to 292±16 g on day 119 (P<0.00001). This resulted ina 1-week increase in weight of 23.2±9.5 g from day 112 to 119, ascompared to 5.1±5.4 g in the preceding week (P<0.00001). Despitecontinuing on this high-fat diet, by day 182 the rats weighed 296±22 g,which was not statistically different from their weights 1 week afterstarting on high-fat chow (P=0.39).

In addition to the weight change seen after switching to high-fat chow,stool M. smithii levels also increased suddenly after the high-fat dietwas implemented (FIG. 10b ). M. smithii levels were 5.6×104±2.8×103cfu/ml which increased by nearly 1 log to 3.0×105±7.0×103 cfu/ml after 1week of high-fat diet (P<0.01) (FIG. 11a ). Like the change in bodyweight, the change in M. smithii occurred in 1 week and did not furtherincrease with additional weeks on high-fat chow (FIG. 11a ).

In another analysis, rats were divided into groups based on those thatgained more or less weight with high-fat. In another analysis, rats weredivided into groups based on those that gained more or less weight withhigh-fat. In this analysis, rats which had>10% weight gain with high-fathad higher stool levels of M. smithii than rats which gained less weight(<10% weight gain) (P=0.08, FIG. 11b ).

M. smithii and Body Weight on Returning to Normal Diet

On returning to normal chow after 10 weeks of high-fat diet on day 182,rats did not experience a reduction in body weight (FIG. 10a ). Asdepicted in this figure, the rat weights remained at a plateau. However,the return to normal chow resulted in a gradual reduction in stool M.smithii levels over time (FIG. 10b ). On day 189, 1 week after cessationof fat and resumption of normal chow, M. smithii levels were3.4×103±8.1×10² cfu/ml, which was significantly reduced from day 154(P<0.001) (FIG. 10b ). Stool M. smithii levels continued to decline inrats continued on normal chow to the end of the study (2.0×102±2.0×102cfu/ml) (P<0.05) (FIG. 10b ).

Randomizing Back to High-Fat Diet a Second Time

In the final phase of the study, rats were randomized into two groups(10 rats returned to high-fat chow and the other 10 continued on normalchow). While FIG. 10a suggests that returning to high-fat chow didfurther increase body weight compared to continuing on normal chow, thedifferences in weights between the two groups (high-fat vs. normal chow)did not reach statistical significance for any timepoint. However, the10 rats returned to high-fat chow exhibited an increase in averageweight from 292±16 g to 319±26 g, which was significant (P<0.001, FIG.10a ). The return to high-fat chow also resulted in an increase in M.smithii levels in these animals (P=0.039, FIG. 11c ).

Bacteria and M. smithii Levels by Bowel Segment Post-Mortem

Following euthanasia on day 287 post-inoculation, sections of the leftcolon, cecum, ileum, jejunum, and duodenum were resected from each rat,and DNA was extracted from luminal contents of each segment. qPCR withM. smithii-specific and universal bacterial primers was used todetermine the levels of M. smithii and total bacteria, respectively.Surprisingly, the highest levels of M. smithii were found in the smallintestine, and were most elevated in the ileum (FIG. 12a ). In contrast,total bacterial levels were lowest in the small intestine, and highestin the cecum and left colon (FIG. 12b ). When the levels of M. smithiiin each bowel segment were compared for rats switched to a high-fat dietin the final phase of the study vs. those maintained on normal chow,higher M. smithii levels were identified in all bowel segments of ratsswitched to a high-fat diet (FIG. 13a ). However, only the duodenum,ileum, and cecum reached statistical significance. In contrast, nosignificant differences in total bacterial levels were identifiedbetween rats switched to a high-fat diet compared to those maintained onnormal chow in any bowel segment (FIG. 13b ).

Correlation Between Extent of Bowel Colonization with M. smithii andWeight in Rats

The final comparison was to examine the distribution of M. smithii inthe GI tract as a determinant of body weight. Although not statisticallysignificant, rats with the greatest extent of M. smithii colonization(i.e., those with no uncolonized bowel segments) had higher weights thanthose with less widespread M. smithii colonization (i.e., those with oneor more uncolonized bowel segments), irrespective of whether or not theywere on high-fat chow in the final phase of the study (FIG. 14). Thelowest body weight of all rats was recorded for a rat on high-fat chowthat had three bowel segments out of five lacking M. smithiicolonization.

Example 3

Study Population

Consecutive subjects presenting for lactulose breath testing wereeligible for participation. Exclusion criteria were based on the abilityto safely perform bioimpedance anthropometric measurements, and pregnantwomen and those with cardiac pacing/defibrillation devices wereexcluded. All subjects provided informed consent prior to participatingin the study. The study was approved by Institutional Review Board atCedars-Sinai Medical Center (Los Angeles, Calif.).

Questionnaire

Subjects completed a demographic and medical questionnaire and a bowelsymptom questionnaire (B12) rating their last 7 days of intestinalcomplaints (bloating, diarrhea, constipation, and abdominal pain) on avisual analog scale from 0 to 100 mm, 100 being the most severe.

Lactulose Breath Test

Subjects presented to the medical center, having fasted for 12 hours asdescribed previously (B13). Breath samples were collected in a dual-bagsystem (Quintron Instrument Co, Milwaukee, Wis.). After an initialbreath collection, subjects ingested 10 g of lactulose syrup and then250 mL of water. Breath samples were collected every 15 minutes for 2hours and analyzed using the Breath-tracker-gas chromatograph (QuintronInstrument Co). Outputs included hydrogen, methane, and carbon dioxide.Hydrogen and methane were corrected for carbon dioxide to standardize toalveolar gas levels and reported in parts per million (ppm). Subjectswith methane 3 ppm or greater were considered methane positive, asdescribed previously (B13). Subjects with hydrogen greater than 20 ppmat or before 90 minutes during the test were considered hydrogenpositive.

Anthropometrics

Bioimpedance testing was performed using the In-Body scale (Biospace Co,Ltd, Seoul, Korea), which has been validated in other studies (B12).BMIand percent body fat were determined based on height (measured viastadiometer) and electrical conductance.

Outcome Measures

Subjects were divided into 4 groups: normal (N) (<3 ppm methane and<2 0ppm hydrogen at or before 90 minutes); hydrogen positive only (H+)(<3ppm methane and hydrogen <20 ppm at or before 90 minutes); methanepositive only (M+) (methane 3 ppm and hydrogen <20 ppm at or before 90minutes); and methane and hydrogen positive (M+/H+) (methane≥3 ppm andhydrogen≥20 ppm at or before 90 minutes). Primary outcome measures wereBMI and percent body fat, and primary analyses compared these measuresacross the 4 groups.

Data and Statistical Analysis

Age was compared across the groups by ANOVA and then Dunnett's post hoctest and gender by the Fisher exact test. Visual analog scale scoreswere compared across the groups by the Kruskal-Wallis test because ofnonnormality. BMI and percent body fat were analyzed by analysis ofcovariance (ANCOVA) models. The initial ANCOVA models were 2-wayfactorial models (sex at 2 levels and group at 4 levels) with age as acovariate. Because the gender-by-group interaction was not significant(P=0.28 for BMI and P=0.37 for percent body fat), the interaction termwas dropped in the ANCOVA model for each outcome. Age was significantand was retained in each model. Least squares (adjusted) means were usedto compare the H+/M+ group with each of the other 3 groups. A 2-sidedsignificance level of P=0.05 was used throughout. SAS version 9.2 (SASInstitute, Cary, N.C.) was used for statistical calculations.

Demographics

A total of 792 subjects participated in the study. Subject demographicswere noted and somewhat different between groups (Table 2). Subjects inthe methane-positive only (M+) and methane- and hydrogen-positive(H+/M+) groups were older than those in the normal (N) andhydrogen-positive only (H+) groups. The percent of females was lower inthe H+ and M+ groups. Baseline GI complaints were not different betweengroups, although M+ subjects tended to have a greater degree ofconstipation than other groups (Table 2).

TABLE 2 Demographic Comparison of the Study Cohort Total N H+ M+ H+/M+ PBaseline Variable (n = 792) (n = 343) (n = 320) (n = 101) (n = 28)Value^(a) Demographic Age, 47.3 ± 16.3 46.7 ± 16.2 45.7 ± 15.9 53.2 ±15.5 50.1 ± 19.7 <.001 Gender, % 70.8 75.51 67.5 65.35 71.43 .073 femaleIntestinal Bloating 61.3 ± 28.4 61.3 ± 29.4 61.1 ± 27.8 61.7 ± 27.6 60.8± 26.2 .97 symptoms Abd pain 47.9 ± 31.7 49.8 ± 31.4 46.4 ± 31.6 49.1 ±32.6 39.0 ± 31.4 .30 (mean VAS) Constipation 42.3 ± 35.1 40.4 ± 34.841.8 ± 35.5 47.6 ± 34.8 52.8 ± 33.8 .16 Diarrhea 35.4 ± 33.7 36.2 ± 33.936.0 ± 34.0 31.2 ± 32.9 34.2 ± 30.2 .53 Data are expressed as mean ±SD;^(a)P value is for comparison of differences among the 4 groups.

Body Composition

H+/M+ subjects had a greater BMI than any of the other 3 groups (FIG.15A). Similarly, percent body fat was greatest in the H+/M+ group (FIG.15B). Gender was not significantly different between groups. ANOVAindicated that age differed across the groups. Dunnett's post hoc testindicated that the M+ group was the only group that differedsignificantly from the N group for age. Adjusting for age, BMI was stillsignificantly higher in the H+/M+ group than the other 3 groups (N:24.1±5.2 kg/m²; H+: 24.2±4.5 kg/m²; M+: 24.0±3.75 kg/m²; H+/M+: 26.5±7.1kg/m2, P<0.02 for each comparison). Using a similar analysis, the H+/M+group had a higher percent body fat than the other groups (N:28.3±10.0%; H+: 27.5±9.0%; M+: 28.0±8.9%; H+/M+: 34.1±10.9%) (P<0.001for each comparison).

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Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. It will be understood by those within the art that,in general, terms used herein are generally intended as “open” terms(e.g., the term “including” should be interpreted as “including but notlimited to,” the term “having” should be interpreted as “having atleast,” the term “includes” should be interpreted as “includes but isnot limited to,” etc.).

1-56. (canceled)
 57. A method, comprising: subjecting a biologicalsample from a subject to analysis for methanogen quantity, wherein theanalysis comprises using quantitative polymerase chain reaction (qPCR)or breath test; comparing the methanogen quantity to a reference value:selecting a statin for the subject if the methanogen quantity is higherthan the reference value based on the recognition that the statin isappropriate for subjects who have a methanogen quantity higher than thereference value; and administering an effective amount of the statin tothe subject, wherein the subject has or is suspected to haveconstipation predominant irritable bowel syndrome (C-IBS) caused by orassociated with having a high methanogen quantity.
 58. The method ofclaim 57, further comprising subjecting the biological sample toanalysis for a quantity of a methanogen syntrophic microorganism. 59.The method of claim 58, wherein the methanogen syntrophic microorganismis a hydrogen-producing microorganism.
 60. The method of claim 58,further comprising selecting a therapy to inhibit the growth of themethanogen syntrophic microorganism.
 61. The method of claim 60, furthercomprising administering to the subject the therapy to inhibit thegrowth of the methanogen syntrophic microorganism.
 62. The method ofclaim 57, wherein the methanogen is from the genus Methanobrevibacter.63. The method of claim 62, wherein the Methanobrevibacter isMethanobrevibacter smithii (M. Smithii).
 64. The method of claim 57,wherein the reference value is about 10,000 cfu per ml of the biologicalsample.
 65. The method of claim 57, wherein the statin is selected fromatorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin,rosuvastatin, simvastatin, and combinations thereof.
 66. A method,comprising: subjecting a biological sample from a subject to analysisfor methanogen quantity, wherein the analysis comprises usingquantitative polymerase chain reaction (qPCR) or a breath test;comparing the methanogen quantity to a reference value; selecting astatin for the subject if the methanogen quantity is higher than thereference value based on the recognition that the statin is appropriatefor subjects who have a methanogen quantity higher than the referencevalue; and administering an effective amount of the statin to thesubject, wherein the subject desires a determination of susceptibilityto having constipation predominant irritable bowel syndrome (C-IBS)caused by or associated with having a high methanogen quantity.
 67. Themethod of claim 66, further comprising subjecting the biological sampleto analysis for a quantity of a methanogen syntrophic microorganism. 68.The method of claim 67, wherein the methanogen syntrophic microorganismis a hydrogen-producing microorganism.
 69. The method of claim 67,further comprising selecting a therapy to inhibit the growth of themethanogen syntrophic microorganism.
 70. The method of claim 69, furthercomprising administering to the subject the therapy to inhibit thegrowth of the methanogen syntrophic microorganism.
 71. The method ofclaim 66, wherein the methanogen is from the genus Methanobrevibacter.72. The method of claim 71, wherein the Methanobrevibacter isMethanobrevibacter smithii (M. Smithii).
 73. The method of claim 66,wherein the reference value is about 10,000 cfu per ml of the biologicalsample.
 74. The method of claim 66, wherein the statin is selected fromatorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin,rosuvastatin, simvastatin, and combinations thereof.